Yokukansan Suppresses Gastric Hypersensitivity and Eosinophil-associated Microinflammation in Rats With Functional Dyspepsia.

Background/Aims: Herbal medicine is an important complementary therapy for functional dyspepsia (FD). However, its effect against gastric hypersensitivity in patients with FD has rarely been evaluated. Yokukansan (YKS), a traditional Japanese herbal medicine, is effective against neuropathic and inflammatory pain. This study aims to use a maternal separation (MS) stress-induced FD model to investigate the effects of YKS against gastric hypersensitivity, gastric motility, and duodenal micro-inflammation. Methods: The MS stress model was established by separating newborn Sprague-Dawley rats from their mothers for 2 hours a day from postnatal days 1 to 10. At the age of 7-8 weeks, the rats were treated with YKS at a dose of 5 mL/kg (1 g/kg) for 7 consecutive days. After YKS treatment, electromyographic activity in the acromiotrapezius muscle by gastric distention and the gastric-emptying rate were assessed. Immunohistochemical analysis of eosinophils in the duodenum and phosphorylated extracellular signal-regulated kinase (p-ERK) 1/2 in the spinal cord was performed. Results: YKS treatment suppressed MS stress-induced gastric hypersensitivity and decreased the elevated levels of p-ERK1/2 in the spinal cord. In the gastroduodenal tract, YKS inhibited eosinophil-associated micro-inflammation but did not improve gastric dysmotility. Conclusions: YKS treatment improved gastric hypersensitivity by alleviating eosinophil-associated micro-inflammation in the gastroduodenal tract. This treatment may be considered an effective therapeutic option for epigastric pain and micro-inflammation in patients with FD.

Reactive Oxygen Species Cause Exercise-Induced Angina in a Myocardial Ischaemia-Reperfusion Injury Model.

Percutaneous coronary intervention (PCI) effectively treats obstructive coronary artery syndrome. However, 30-40% patients continue to have angina after a successful PCI, thereby reducing patient satisfaction. The mechanisms underlying persistent angina after revascularisation therapy are still poorly understood; hence, the treatment or guideline for post-PCI angina remains unestablished. Thus, this study aimed to investigate the mechanisms underlying effort angina in animals following myocardial ischaemia-reperfusion (I/R) injury. Phosphorylated extracellular signal-regulated kinase (p-ERK), a marker for painful stimulation-induced neuronal activation, was used for the investigation. After a forced treadmill exercise (FTE), the number of p-ERK-expressing neurons increased in the superficial dorsal horn of the I/R model animals. Moreover, FTE evoked hydrogen peroxide (H2O2) production in the I/R-injured heart, inducing angina through TRPA1 activation on cardiac sensory fibres. Notably, the treatment of a TEMPOL, a reactive oxygen species scavenger, or TRPA1-/- mice successfully alleviated the FTE-induced p-ERK expression in the dorsal horn. The production of H2O2, a reactive oxygen species, through physical exercise contributes to angina development following I/R. Hence, our findings may be useful for understanding and treating angina following revascularisation therapy.

Atractylodin Produces Antinociceptive Effect through a Long-Lasting TRPA1 Channel Activation.

Atractylodin (ATR) is a bioactive component found in dried rhizomes of Atractylodes lancea (AL) De Candolle. Although AL has accumulated empirical evidence for the treatment of pain, the molecular mechanism underlying the anti-pain effect of ATR remains unclear. In this study, we found that ATR increases transient receptor potential ankyrin-1 (TRPA1) single-channel activity in hTRPA1 expressing HEK293 cells. A bath application of ATR produced a long-lasting calcium response, and the response was completely diminished in the dorsal root ganglion neurons of TRPA1 knockout mice. Intraplantar injection of ATR evoked moderate and prolonged nociceptive behavior compared to the injection of allyl isothiocyanate (AITC). Systemic application of ATR inhibited AITC-induced nociceptive responses in a dose-dependent manner. Co-application of ATR and QX-314 increased the noxious heat threshold compared with AITC in vivo. Collectively, we concluded that ATR is a unique agonist of TRPA1 channels, which produces long-lasting channel activation. Our results indicated ATR-mediated anti-nociceptive effect through the desensitization of TRPA1-expressing nociceptors.

The ubiquitin E3 ligase Nedd4-2 relieves mechanical allodynia through the ubiquitination of TRPA1 channel in db/db mice.

Neural precursor cell-expressed developmentally downregulated protein 4-2 (Nedd4-2) is a member of the E3 ubiquitin ligase family that is highly expressed in sensory neurons and involved in pain modulation via downregulation of ion channels in excitable membranes. Ubiquitination involving Nedd4-2 is regulated by adenosine monophosphate-activated protein kinase (AMPK), which is impaired in the dorsal root ganglion (DRG) neurons of db/db mice. AMPK negatively regulates the expression of transient receptor potential ankyrin 1 (TRPA1), a recognised pain sensor expressed on the membrane of DRG neurons, consequently relieving mechanical allodynia in db/db mice. Herein, we studied the involvement of Nedd4-2 in painful diabetic neuropathy and observed that Nedd4-2 negatively regulated diabetic mechanical allodynia. Nedd4-2 was co-expressed with TRPA1 in mouse DRG neurons. Nedd4-2 was involved in TRPA1 ubiquitination, this ubiquitination, as well as Nedd4-2-TRPA1 interaction, was decreased in db/db mice. Moreover, Nedd4-2 levels were decreased in db/db mice, while an abnormal intracellular distribution was observed in short-term high glucose-cultured DRG neurons. AMPK activators not only restored Nedd4-2 distribution but also increased Nedd4-2 expression. These findings demonstrate that Nedd4-2 is a potent regulator of TRPA1 and that the abnormal expression of Nedd4-2 in DRG neurons contributes to diabetic neuropathic pain.

Eosinophil-associated microinflammation in the gastroduodenal tract contributes to gastric hypersensitivity in a rat model of early-life adversity.

Gastric hypersensitivity is a major pathophysiological feature of functional dyspepsia (FD). Recent clinical studies have shown that a large number of patients with FD present with gastroduodenal microinflammation, which may be involved in the pathophysiology of FD. However, no animal model reflecting this clinical characteristic has been established. The underlying mechanism between microinflammation and FD remains unknown. In this study, using a maternal separation (MS)-induced FD model, we aimed to reproduce the gastroduodenal microinflammation and reveal the interaction between gastroduodenal microinflammation and gastric hypersensitivity. The MS model was established by separating newborn Sprague-Dawley rats for 2 h a day from postnatal day 1 to day 10. At 7-8 wk of age, electromyography was used to determine the visceromotor response to gastric distention (GD) and immunohistochemistry was performed to detect distension-associated neuronal activation as well as immunohistological changes. Our results demonstrated that MS-induced FD rats underwent gastric hypersensitivity with GD at 60 and 80 mmHg, which are related to increased p-ERK1/2 expression in the dorsal horn of T9-T10 spinal cords. Eosinophils, but not mast cells, were significantly increased in the gastroduodenal tract, and the coexpression rate of CD11b and major basic protein significantly increased in MS rats. Treatment with dexamethasone reversed gastric hypersensitivity in MS-induced FD rats by inhibiting eosinophil infiltration. These findings indicated that neonatal MS stress induces eosinophil-associated gastroduodenal microinflammation and gastric hypersensitivity in adulthood in rats. Microinflammation contributes to gastric hypersensitivity; therefore, anti-inflammatory therapy may be effective in treating patients with FD with gastroduodenal microinflammation.NEW & NOTEWORTHY We showed for the first time that neonatal MS stress-induced FD rats undergo gastroduodenal eosinophil-associated microinflammation in adulthood. Suppression of microinflammation attenuated gastric hypersensitivity in MS rats. These findings established a functional link between microinflammation and gastric hypersensitivity, which may provide a potential clue for the clinical treatment of FD.

Daikenchuto attenuates visceral pain and suppresses eosinophil infiltration in inflammatory bowel disease in murine models.

BACKGROUND AND AIM: Daikenchuto (DKT), a traditional Japanese formula, comprises four herbal medicines and is used for abdominal pain. Inflammatory bowel disease (IBD) includes ulcerative colitis (UC) and Crohn's disease (CD) and is characterized by colonic inflammation and chronic abdominal pain. The present study aimed to investigate whether DKT suppresses colonic hypersensitivity and inflammation associated with IBD in animal models. METHODS: Sprague-Dawley rats were administered 4% sodium dextran sulfate (DSS) or trinitrobenzene sulfate (TNBS) in the colon to establish UC or CD models, respectively. DKT and 5-aminosalicylic acid (5-ASA) were administered orally once a day from Days 3 to 7 after induction of colitis. On Day 7, visceral pain and inflammation were evaluated by measuring the visceromotor response (VMR) to colorectal distention (CRD) and inflammatory indicators, including histological score, length of leukocyte infiltration, MPO activity, and eosinophil count. RESULTS: DSS and TNBS increased VMR to CRD and the inflammation indicators. DKT, but not 5-ASA, suppressed the VMR to CRD in DSS- and TNBS-treated rats. DKT and 5-ASA decreased the eosinophil count in both IBD models. In DSS-treated rats, 5-ASA, but not DKT, suppressed the MPO activity. In TNBS-treated rats, neither 5-ASA nor DKT suppressed MPO activity. CONCLUSION: These results suggest that DKT is beneficial for abdominal pain associated with IBD. The anti-inflammatory effect of DKT on IBD may involve inhibition of eosinophils. The mechanism of anti-inflammatory effect of DKT partially differs from that of 5-ASA. Coapplication of DKT and conventional medicine may produce a positive synergy effect for IBD treatment.

TRPA1-expressing lamina propria mesenchymal cells regulate colonic motility.

The physiological process of defecation is directly controlled by colorectal motility. The transient receptor potential ankyrin 1 (TRPA1) channel is expressed in small intestine enterochromaffin cells and is involved in gastrointestinal motility via serotonin release. In the colorectum, however, enterochromaffin cell localization is largely distinct from that in the small intestine. Here, we investigated the role of lower gastrointestinal tract TRPA1 in modulating colorectal motility. We found that in colonic tissue, TRPA1 is predominantly expressed in mesenchymal cells of the lamina propria, which are clearly distinct from those in the small intestine. These cells coexpressed COX1 and microsomal prostaglandin E synthase-1. Intracolonic administration of TRPA1 agonists induced colonic contraction, which was suppressed by a prostaglandin E2 (PGE2) receptor 1 antagonist. TRPA1 activation induced calcium influx and PGE2 release from cultured human fibroblastic cells. In dextran sulfate sodium-treated animals, both TRPA1 and its endogenous agonist were dramatically increased in the colonic lamina propria, accompanied by abnormal colorectal contractions. Abnormal colorectal contractions were significantly prevented by pharmacological and genetic inhibition of TRPA1. In conclusion, in the lower gastrointestinal tract, mesenchymal TRPA1 activation results in PGE2 release and consequently promotes colorectal contraction, representing what we believe is a novel physiological and inflammatory bowel disease-associated mechanism of gastrointestinal motility.

Negative Regulation of TRPA1 by AMPK in Primary Sensory Neurons as a Potential Mechanism of Painful Diabetic Neuropathy.

AMPK is a widely expressed intracellular energy sensor that monitors and modulates energy expenditure. Transient receptor potential ankyrin 1 (TRPA1) channel is a widely recognized chemical and thermal sensor that plays vital roles in pain transduction. In this study, we discovered a functional link between AMPK and TRPA1 in dorsal root ganglion (DRG) neurons, in which AMPK activation rapidly resulted in downregulation of membrane-associated TRPA1 and its channel activity within minutes. Treatment with two AMPK activators, metformin or AICAR, inhibited TRPA1 activity in DRG neurons by decreasing the amount of membrane-associated TRPA1. Metformin induced a dose-dependent inhibition of TRPA1-mediated calcium influx. Conversely, in diabetic db/db mice, AMPK activity was impaired in DRG neurons, and this was associated with a concomitant increase in membrane-associated TRPA1 and mechanical allodynia. Notably, these molecular and behavioral changes were normalized following treatment with AMPK activators. Moreover, high-glucose exposure decreased activated AMPK levels and increased agonist-evoked TRPA1 currents in cultured DRG neurons, and these effects were prevented by treatment with AMPK activators. Our results identify AMPK as a previously unknown regulator of TRPA1 channels. AMPK modulation of TRPA1 could thus serve as an underlying mechanism and potential therapeutic molecular target in painful diabetic neuropathy.

Synthesis of resveratrol derivatives as new analgesic drugs through desensitization of the TRPA1 receptor.

A series of 31 resveratrol derivatives was designed, synthesized and evaluated for activation and inhibition of the TRPA1 channel. Most acted as activators and desensitizers of TRPA1 channels like resveratrol or allyl isothiocyanate (AITC). Compound 4z (HUHS029) exhibited higher inhibitory activity than resveratrol with an IC50 value of 16.1µM. The activity of 4z on TRPA1 was confirmed in TRPA1-expressing HEK293 cells, as well as in rat dorsal root ganglia neurons by a whole cell patch clamp recording. Furthermore, pretreatment with 4z exhibited an analgesic effect on AITC-evoked TRPA1-related pain behavior in vivo.

Partial Activation and Inhibition of TRPV1 Channels by Evodiamine and Rutaecarpine, Two Major Components of the Fruits of Evodia rutaecarpa.

Evodiamine (1) and rutaecarpine (2) are the two major components of Evodia rutaecarpa, which has long been used in traditional medicine for the treatment of many diseases. Using transient receptor potential vanilloid 1 (TRPV1)-expressing HEK293 cells and patch-clamp recording, the inhibitory actions of 1 and 2 against TRPV1 channels were investigated. The effects of these compounds against capsaicin- or proton-activated TRPV1 activities were evaluated. The results showed that, although 1 and 2 can activate TRPV1, the maximum response was 3.5- or 9-fold lower than that of capsaicin, respectively, suggesting partial agonism. In comparison to capsaicin, coadministration of 1 and capsaicin increased the half-maximal effective concentration (EC50) of capsaicin-activated TRPV1 currents as shown by a right shift in the dose-response curve, whereas coadministration of 1 with protons failed to inhibit the proton-induced current. Moreover, preadministration of 1, but not 2, inhibited both capsaicin- and proton-induced TRPV1 currents, which might involve channel desensitization. Taken together, 1 and 2 may share the same binding site with capsaicin and act as partial agonists (antagonists) of TRPV1. Evodiamine (1), but not rutaecarpine (2), can desensitize or competitively inhibit the activity of TRPV1.

Elevated H2 O2 levels in trinitrobenzene sulfate-induced colitis rats contributes to visceral hyperalgesia through interaction with the transient receptor potential ankyrin 1 cation channel.

BACKGROUND AND AIM: Inflammatory bowel disease is associated with chronic abdominal pain. Transient receptor potential ankyrin 1 (TRPA1) is a well-known pain sensor expressed in primary sensory neurons. Recent studies indicate that reactive oxygen species such as hydrogen peroxide (H2 O2 ) may activate TRPA1. METHODS: Colonic inflammation was induced by intra-colonic administration of trinitrobenzene sulfate (TNBS) in adult male Sprague-Dawley rats. Visceromotor response (VMR) to colorectal distention (CRD) was recorded to evaluate the visceral hyperalgesia. Rats were sacrificed 1 day after treatment with saline or TNBS; colonic tissues from the inflamed region were removed and then processed to assess the H2 O2 content. H2 O2 scavenger N-acetyl-l-cysteine or a TRPA1 antagonist, HC-030031, was intravenously administrated to the TNBS-treated rats or saline-treated rats. In a parallel experiment, intra-colonic H2 O2 -induced visceral hyperalgesia in naïve rats and the effect of intravenous HC-030031 were measured based on the VMR to CRD. RESULTS: Trinitrobenzene sulfate treatment resulted in significant increase in VMR to CRD at day 1. The H2 O2 content in the inflamed region of the colon in TNBS-treated rats was significantly higher than that of saline-treated rats. N-acetyl-l-cysteine or HC-030031 significantly suppressed the enhanced VMR in TNBS-treated rats while saline-treated rats remained unaffected. Moreover, blockade of TRPA1 activation by HC-030031 significantly reversed the exogenous H2 O2 -induced visceral hyperalgesia. CONCLUSION: These results suggest that H2 O2 content of the colonic tissue is increased in the early stage of TNBS-induced colitis. The increased H2 O2 content may contribute to the visceral hyperalgesia by activating TRPA1.

Evodiamine suppresses capsaicin-induced thermal hyperalgesia through activation and subsequent desensitization of the transient receptor potential V1 channels.

Evodiae fructus (EF), a fruit of Evodia rutaecarpa Bentham, has long been used as an analgesic drug in traditional Chinese and Japanese medicine. However, the underlying molecular mechanism of its pharmacological action is unclear. Here, using calcium imaging, whole-cell patch-clamp recording, and behavioral analysis, we investigated the pharmacological action of EF and its principal compound, evodiamine, on the transient receptor potential (TRP) V1 channels. Dorsal root ganglion (DRG) neurons and TRPV1- or TRPA1-transfected human embryonic kidney-derived (HEK) 293 cells were used for calcium imaging or whole-cell patch-clamp recording. Twenty male adult Sprague-Dawley rats were used for the capsaicin-induced thermal hyperalgesia behavioral analyses. We found that evodiamine induced significant increases in intracellular calcium and robust inward currents in a subpopulation of isolated rat DRG neurons, most of which were also sensitive to capsaicin. The effect of evodiamine was completely blocked by capsazepine, a competitive antagonist of TRPV1. Evodiamine induced significant inward currents in TRPV1-, but not TRPA1-transfected HEK293 cells. Pretreatment with evodiamine reduced capsaicin-induced currents significantly. Furthermore, the in vivo pre-treatment of evodiamine suppressed thermal hyperalgesia induced by intraplantar injection of capsaicin in rats. These results identify that the analgesic effect of EF and evodiamine may be due to the activation and subsequent desensitization of TRPV1 in sensory neurons.

Role of neuropsin in parvalbumin immunoreactivity changes in hippocampal basket terminals of mice reared in various environments.

In vitro approaches have suggested that neuropsin (or kallikrein 8/KLK8), which controls gamma-aminobutyric acid (GABA) neurotransmission through neuregulin-1 (NRG-1) and its receptor (ErbB4), is involved in neural plasticity (Tamura et al., 2012, 2013). In the present study, we examined whether parvalbumin (PV)-positive neuronal networks, the majority of which are ErbB4-positive GABAergic interneurons, are controlled by neuropsin in tranquil and stimulated voluntarily behaving mice. Parvalbumin-immunoreactive fibers surrounding hippocampal pyramidal and granular neurons in mice reared in their home cage were decreased in neuropsin-deficient mice, suggesting that neuropsin controls PV immunoreactivity. One- or two-week exposures of wild mice to novel environments, in which they could behave freely and run voluntarily in a wheel resulted in a marked upregulation of both neuropsin mRNA and protein in the hippocampus. To elucidate the functional relevance of the increase in neuropsin during exposure to a rich environment, the intensities of PV-immunoreactive fibers were compared between neuropsin-deficient and wild-type (WT) mice under environmental stimuli. When mice were transferred into novel cages (large cages with toys), the intensity of PV-immunoreactive fibers increased in WT mice and neuropsin-deficient mice. Therefore, behavioral stimuli control a neuropsin-independent form of PV immunoreactivity. However, the neuropsin-dependent part of the change in PV-immunoreactive fibers may occur in the stimulated hippocampus because increased levels of neuropsin continued during these enriched conditions.

Xilei san ameliorates experimental colitis in rats by selectively degrading proinflammatory mediators and promoting mucosal repair.

Xilei san (XLS), a herbal preparation widely used in China for erosive and ulcerative diseases, has been shown to be effective in ulcerative colitis (UC). The present experiments were conducted to assess its efficacy and determine its mechanism of action in a rat model that resembles human UC. The model was induced by adding 4% dextran sulfate sodium (DSS) to the rats' drinking water for 7 days. XLS was administered daily by retention enema from day 2 to day 7; the rats were sacrificed on day 8. The colon tissues were obtained for further experiments. A histological damage score and the activity of tissue myeloperoxidase were used to evaluate the severity of the colitis. The colonic cytokine levels were detected in a suspension array, and epithelial proliferation was assessed using Ki-67 immunohistochemistry. Intrarectal administration of XLS attenuated the DSS-induced colitis, as evidenced by a reduction in both the histological damage score and myeloperoxidase activity. It also decreased the levels of proinflammatory cytokines, but increased the mucosal repair-related cytokines. In addition, the epithelial Ki-67 expression was upregulated by XLS. These results suggest that XLS attenuates DSS-induced colitis by degrading proinflammatory mediators and promoting mucosal repair. XLS could be a potential topical treatment for human UC.

Etodolac activates and desensitizes transient receptor potential ankyrin 1.

The transient receptor potential ankyrin 1 (TRPA1) channel is well known as a sensor to environmental irritant compounds, cold, and endogenous proalgesic agents. TRPA1 is expressed on sensory neurons and is involved in pain modulation. Etodolac is a cyclooxygenase (COX)-2 inhibitor that belongs to the class of nonsteroidal anti-inflammatory drugs (NSAIDs). A recent study indicates that etodolac inhibits allyl isothiocyanate (AITC)-induced calcium influx in heterologous HEK293 cells and sensory neurons. To examine whether and how etodolac modulates the TRPA1 channels, we applied etodolac to TRPA1-transfected HEK293 cells or rat dorsal root ganglion (DRG) neurons and recorded the currents using the whole-cell patch clamp technique. We found that etodolac at higher doses could activate and then desensitize TRPA1 channels in heterologous expressing HEK293 cells as well as in DRG neurons. The etodolac-induced currents were significantly attenuated in cysteine residues mutated human TRPA1-transfected HEK293 cells. Interestingly, application of etodolac at drug plasma levels in clinical usage did not induce significant TRPA1 currents but reduced the subsequent AITC-induced currents to 25% in HEK293 cells expressing TRPA1. Moreover, no modulatory effect of etodolac on TRPA1 was detected in the cysteine mutant cells. These data indicate a novel mechanism of the anti-inflammatory and analgesic clinical effects of etodolac, which may be involved with its direct activation and the subsequent desensitization of TRPA1 through the covalent modification of cysteine residues.

Modulation of TRP channels by resveratrol and other stilbenoids.

BACKGROUND: Resveratrol (3,5,4' - trihydroxy-trans-stilbene), a widely distributed natural stilbenoid, was proposed to account for the unique effects of red wine on life span and health. It has been reported to possess various biological and pharmacological activities, such as anti-oxidant, anti-inflammatory, and anti-carcinogenic effects. Here, using whole-cell patch-clamp techniques and behavioral analyses, we investigated whether resveratrol and other stilbenoids can modulate TRP channels in sensory neurons in vitro, and have analgesic effects in vivo. RESULTS: We found that resveratrol dose-dependently suppressed the allyl isothiocyanate (AITC)-induced currents (I AITC) in HEK293 cells that express TRPA1, as well as in rat dorsal root ganglion (DRG) neurons. Instead, pinosylvin methyl ether (PME), another derivate of stilbene which has a similar structure to resveratrol, dose-dependently blocked the capsaicin-induced currents (I CAP) in HEK293 cells that express TRPV1 as well as in DRG neurons. Interestingly, resveratrol had no inhibitory effect on the I CAP, and PME had no effect on the I AITC. Otherwise, trans-stilbene showed no any effect on I AITC or I CAP. The concentration response curve of AITC showed that resveratrol inhibited the action of TRPA1 not by changing the EC50, but by suppressing the AITC-induced maximum response. By contrast, the inhibition of TRPV1 by PME did not change the capsaicin-induced maximum response but did cause a right shift of the EC50. Moreover, pre-administration of resveratrol suppressed intraplantar injections of AITC-evoked nocifensive behaviors, as well as that PME suppressed capsaicin-evoked one. CONCLUSIONS: These data suggest that resveratrol and other stilbenoids may have an inhibitory effect on TRP channels. In addition, these stilbenoids modulate TRP channel activity in different ways.

Potentiation of the P2X3 ATP receptor by PAR-2 in rat dorsal root ganglia neurons, through protein kinase-dependent mechanisms, contributes to inflammatory pain.

Proinflammatory agents trypsin and mast cell tryptase cleave and activate protease-activated receptor-2 (PAR-2), which is expressed on sensory nerves and causes neurogenic inflammation. P2X3 is a subtype of the ionotropic receptors for adenosine 5'-triphosphate (ATP), and is mainly localized on nociceptors. Here, we show that a functional interaction of the PAR-2 and P2X3 in primary sensory neurons could contribute to inflammatory pain. PAR-2 activation increased the P2X3 currents evoked by α, ß, methylene ATP in dorsal root ganglia (DRG) neurons. Application of inhibitors of either protein kinase C (PKC) or protein kinase A (PKA) suppressed this potentiation. Consistent with this, a PKC or PKA activator mimicked the PAR-2-mediated potentiation of P2X3 currents. In the in vitro phosphorylation experiments, application of a PAR-2 agonist failed to establish phosphorylation of the P2X3 either on the serine or the threonine site. In contrast, application of a PAR-2 agonist induced trafficking of the P2X3 from the cytoplasm to the plasma membrane. These findings indicate that PAR-2 agonists may potentiate the P2X3, and the mechanism of this potentiation is likely to be a result of translocation, but not phosphorylation. The functional interaction between P2X3 and PAR-2 was also confirmed by detection of the α, ß, methylene-ATP-evoked extracellular signal-regulated kinases (ERK) activation, a marker of neuronal signal transduction in DRG neurons, and pain behavior. These results demonstrate a functional interaction of the protease signal with the ATP signal, and a novel mechanism through which protease released in response to tissue inflammation might trigger the sensation to pain through P2X3 activation.