A new rat model for thrombus-induced ischemic pain (TIIP); development of bilateral mechanical allodynia

Patients with peripheral arterial disease (PAD) commonly suffer from ischemic pain associated with severe thrombosis. However, the pathophysiology of peripheral ischemic pain is not fully understood due to the lack of an adequate animal model. In this study, we developed a new rodent model of thrombus-induced ischemic pain (TIIP) to investigate the neuronal mechanisms underlying ischemic pain. Ischemia was induced by application of 20% FeCl(2) onto the surface of the femoral artery for 20min. Induction of peripheral ischemia was confirmed by measurement of the concentration of Evans blue and by increases in the ischemia-specific markers, hypoxia-inducible factor-1 alpha and vascular endothelial growth factor in the ipsilateral plantar muscles. Ischemic pain, as indicated by the presence of mechanical allodynia, developed bilaterally and peaked at days 3-9 post-FeCl(2) application and gradually decreased through day 31. Systemic heparin pretreatment dose dependently suppressed ischemic pain, suggesting that thrombosis-induced ischemia might be a key factor in TIIP. Intraplantar injection of BMS-182874, an ET(A) (endothelin-A) receptor antagonist, at day 3 selectively blocked ipsilateral pain, indicating that ET(A) receptor activity mediated TIIP. Spinal GFAP expression was significantly increased by FeCl(2) and intrathecal injection of carbenoxolone (an astrocyte gap junction decoupler) at day 3 significantly reduced TIIP, suggesting that spinal astrocyte activation plays an important role. However, the anti-inflammatory agent, ibuprofen, did not affect TIIP. In conclusion, we have developed a novel animal model of TIIP that should be useful in investigating the pathophysiological mechanisms that underlie human peripheral ischemic pain.     

Electrophysiological and in vivo characterization of A-317567, a novel blocker of acid sensing ion channels

Acid Sensing Ion Channels (ASICs) are a group of sodium-selective ion channels that are activated by low extracellular pH. The role of ASIC in disease states remains unclear partly due to the lack of selective pharmacological agents. In this report, we describe the effects of A-317567, a novel non-amiloride blocker, on three distinct types of native ASIC currents evoked in acutely dissociated adult rat dorsal root ganglion (DRG) neurons. A-317567 produced concentration-dependent inhibition of all pH 4.5-evoked ASIC currents with an IC50 ranging between 2 and 30muM, depending upon the type of ASIC current activated. Unlike amiloride, A-317567 equipotently blocked the sustained phase of ASIC3-like current, a biphasic current akin to cloned ASIC3, which is predominant in DRG. When evaluated in the rat Complete Freud's Adjuvant (CFA)-induced inflammatory thermal hyperalgesia model, A-317567 was fully efficacious at a dose 10-fold lower than amiloride. A-317567 was also potent and fully efficacious when tested in the skin incision model of post-operative pain. A-317567 was entirely devoid of any diuresis or natriuresis activity and showed minimal brain penetration. In summary, A-317567 is the first reported small molecule non-amiloride blocker of ASIC that is peripherally active and is more potent than amiloride in vitro and in vivo pain models. The discovery of A-317567 will greatly help to enhance our understanding of the physiological and pathophysiological role of ASICs.     

Mice lacking acid‐sensing ion channels (ASIC) 1 or 2, but not ASIC3, show increased pain behaviour in the formalin test

Extracellular acidification is a component of the inflammatory process and may be a factor driving the pain accompanying it. Acid-sensing ion channels (ASICs) are neuronal proton sensors and evidence suggests they are involved in signalling inflammatory pain. The aims of this study were to (1) clarify the role of ASICs in nociception and (2) confirm their involvement in inflammatory pain and determine whether this was subunit specific.This was achieved by (1) direct comparison of the sensitivity of ASIC1, ASIC2, ASIC3 and TRPV1 knockout mice versus wildtype littermates to acute thermal and mechanical noxious stimuli and (2) studying the behavioural responses of each transgenic strain to hind paw inflammation with either complete Freund’s adjuvant (CFA) or formalin.Naïve ASIC1^?/? and ASIC2^?/? mice responded normally to acute noxious stimuli, whereas ASIC3^?/? mice were hypersensitive to high intensity thermal stimuli. CFA injection decreased mechanical and thermal withdrawal thresholds for up to 8 days. ASIC2^?/? mice had increased mechanical sensitivity on day 1 post-CFA compared to wildtype controls. TRPV1^?/? mice had significantly reduced thermal, but not mechanical, hyperalgesia on all days after inflammation. Following formalin injection, ASIC1^?/? and ASIC2^?/?, but not ASIC3^?/? or TRPV1^?/?, mice showed enhanced pain behaviour, predominantly in the second phase of the test.These data suggest that whilst ASICs may play a role in mediating inflammatory pain, this role is likely to be modulatory and strongly dependent on channel subtype.     

Upregulations of P2X3 and ASIC3 involve in hyperalgesia induced by cisplatin administration in rats

The role of ion channels expressed in sensory neurons on mechanical and thermal hyperalgesia was examined in a rat model of cisplatin-induced peripheral neuropathy. The rats were injected with 3 mg/kg of cisplatin intraperitoneally once per week for five consecutive weeks. The von Frey test, pin-prick test and plantar test were performed to examine any noxious sensitivity of the skin. The Randall–Selitto test of the gastrocnemius muscle (GM) and the measurement of grip forces were performed to quantify muscle hyperalgesia. Coordination/motor was assessed by Rota-rod testing. Expressions of the ion channels TRPV1, TRPV2, P2X₃ and ASIC3 were examined in dorsal root ganglion (DRG) neurons and the muscle afferent neurons innervating GM. Effects of antagonists against either P2X₃ or ASICs on behavioral responses were evaluated. Mechanical hyperalgesia and allodynia of both skin and muscle were observed in cisplatin-treated animals. Expressions of TRPV2, P2X₃, and ASIC3 increased in all DRG neurons. In addition, expressions of P2X₃ and ASIC3 also increased in muscle afferent neurons in DRGs. Antagonists against P2X_3,2/3 and ASICs showed a suppressive effect on both skin and muscle hyperalgesia induced by cisplatin administration. Upregulation of TRPV2, P2X₃, and ASIC3 may play important roles in the mechanical hyperalgesia induced by cisplatin. Furthermore, cisplatin treatment also induced muscle hyperalgesia in muscle afferent neurons in connection with the upregulation of P2X₃ and ASIC3.     

AMG 9810 [(E)-3-(4-t-butylphenyl)-N-(2,3-dihydrobenzo[b][1,4] dioxin-6-yl)acrylamide], a novel vanilloid receptor 1 (TRPV1) antagonist with antihyperalgesic properties

The vanilloid receptor 1 (VR1 or TRPV1) is a membrane-bound, nonselective cation channel expressed by peripheral sensory neurons. TRPV1 antagonists produce antihyperalgesic effects in animal models of inflammatory and neuropathic pain. Here, we describe the in vitro and in vivo pharmacology of a novel TRPV1 antagonist, AMG 9810, (E)-3-(4-t-butylphenyl)-N-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)acrylamide. AMG 9810 is a competitive antagonist of capsaicin activation (IC50 value for human TRPV1, 24.5 +/- 15.7 nM; rat TRPV1, 85.6 +/- 39.4 nM) and blocks all known modes of TRPV1 activation, including protons (IC50 value for rat TRPV1, 294 +/- 192 nM; human TRPV1, 92.7 +/- 72.8 nM), heat (IC50 value for rat TRPV1, 21 +/- 17 nM; human TRPV1, 15.8 +/- 10.8 nM), and endogenous ligands, such as anandamide, N-arachidonyl dopamine, and oleoyldopamine. AMG 9810 blocks capsaicin-evoked depolarization and calcitonin gene-related peptide release in cultures of rat dorsal root ganglion primary neurons. Screening of AMG 9810 against a panel of G protein-coupled receptors and ion channels indicated selectivity toward TRPV1. In vivo, AMG 9810 is effective at preventing capsaicin-induced eye wiping in a dose-dependent manner, and it reverses thermal and mechanical hyperalgesia in a model of inflammatory pain induced by intraplantar injection of complete Freund's adjuvant. At effective doses, AMG 9810 did not show any significant effects on motor function, as measured by open field locomotor activity and motor coordination tests. AMG 9810 is the first cinnamide TRPV1 antagonist reported to block capsaicin-induced eye wiping behavior and reverse hyperalgesia in an animal model of inflammatory pain.     

sigma-1 receptor modulation of acid-sensing ion channel a (ASIC1a) and ASIC1a-induced Ca2+ influx in rat cortical neurons

Acid-sensing ion channels (ASICs) are proton-gated cation channels found in peripheral and central nervous system neurons. The ASIC1a subtype, which has high Ca2+ permeability, is activated by ischemia-induced acidosis and contributes to the neuronal loss that accompanies ischemic stroke. Our laboratory has shown that activation of sigma receptors depresses ion channel activity and [Ca2+](i) dysregulation during ischemia, which enhances neuronal survival. Whole-cell patch-clamp electrophysiology and fluorometric Ca2+ imaging were used to determine whether sigma receptors regulate the function of ASIC in cultured rat cortical neurons. Bath application of the selective ASIC1a blocker, psalmotoxin1, decreased proton-evoked [Ca2+](i) transients and peak membrane currents, suggesting the presence of homomeric ASIC1a channels. The pan-selective sigma-1/sigma-2 receptor agonists, 1,3-di-o-tolyl-guanidine (100 microM) and opipramol (10 microM), reversibly decreased acid-induced elevations in [Ca2+](i) and membrane currents. Pharmacological experiments using sigma receptor-subtype-specific agonists demonstrated that sigma-1, but not sigma-2, receptors inhibit ASIC1a-induced Ca2+ elevations. These results were confirmed using the irreversible sigma receptor antagonist metaphit (50 microM) and the selective sigma-1 antagonist BD1063 (10 nM), which obtunded the inhibitory effects of the sigma-1 agonist, carbetapentane. Activation of ASIC1a was shown to stimulate downstream Ca2+ influx pathways, specifically N-methyl-D-aspartate and (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid/kainate receptors and voltage-gated Ca2+ channels. These subsequent Ca2+ influxes were also inhibited upon activation of sigma-1 receptors. These findings demonstrate that sigma-1 receptor stimulation inhibits ASIC1a-mediated membrane currents and consequent intracellular Ca2+ accumulation. The ability to control ionic imbalances and Ca2+ dysregulation evoked by ASIC1a activation makes sigma receptors an attractive target for ischemic stroke therapy.     

<Emphasis Type="Italic">Acid-Sensing Ion Channels</Emphasis> : des canaux ioniques sensibles au pH et aux lipides dans la douleur

Acid-Sensing Ion Channels (ASICs) are excitatory cationic channels expressed all along the pain neuraxis. They mainly carry Na⁺ ions and generate depolarizing currents in neurons following acidification of the extracellular medium. Different ASIC subunits (ASIC1-4) have been identified in mammals, and a functional channel is formed by the association of three of these subunits, leading to homomeric and heteromeric channels. An increasing number of data obtained by combining pharmacological and genetic tools strongly argues for an involvement of ASICs in pain. From a general point of view, pharmacological inhibition of ASICs, at different levels of the nervous system, produces strong analgesic effects in different animal models of pain, whereas injections of compounds able to activate ASICs generate pain behaviors. ASIC1 and ASIC3 participate in nociception and peripheral sensitization of primary sensory neurons, where ASIC3, which is also activated by lipids, seems to play a major role in inflammatory pain, pruritis and pain arising from deep tissues such as muscle and joint. In the central nervous system, ASIC channels made of ASIC1a and ASIC2 subunits are predominant, and their inhibition at spinal and supra-spinal levels induces powerful analgesia in inflammatory and neuropathic pain models. Together, the data summarized in this review illustrate the therapeutical potential of ASIC channels for pain control.     

Amiloride-blockable acid-sensing ion channels are leading acid sensors expressed in human nociceptors

Many painful inflammatory and ischemic conditions such as rheumatoid arthritis, cardiac ischemia, and exhausted skeletal muscles are accompanied by local tissue acidosis. In such acidotic states, extracellular protons provoke the pain by opening cation channels in nociceptors. It is generally believed that a vanilloid receptor subtype-1 (VR1) and an acid-sensing ion channel (ASIC) mediate the greater part of acid-induced nociception in mammals. Here we provide evidence for the involvement of both channels in acid-evoked pain in humans and show their relative contributions to the nociception. In our psychophysical experiments, direct infusion of acidic solutions (pH > or = 6.0) into human skin caused localized pain, which was blocked by amiloride, an inhibitor of ASICs, but not by capsazepine, an inhibitor of VR1. Under more severe acidification (pH 5.0) amiloride was less effective in reducing acid-evoked pain. In addition, capsazepine had a partial blocking effect under these conditions. Amiloride itself neither blocked capsaicin-evoked localized pain in human skin nor inhibited proton-induced currents in VR1-expressing Xenopus oocytes. Our results suggest that ASICs are leading acid sensors in human nociceptors and that VR1 participates in the nociception mainly under extremely acidic conditions.     

Evidence for the involvement of endogenous ATP and P2X receptors in TMJ pain.

Evidence is accumulating which supports a role for ATP in the initiation of pain by acting on P2X receptors expressed on nociceptive afferent nerve terminals. To investigate whether these receptors play a role in temporomandibular (TMJ) pain, we studied the presence of functional P2X receptors in rat TMJ by examining the nociceptive behavioral response to the application of the selective P2X receptor agonist alpha,beta-methylene ATP (alpha,beta-meATP) into the TMJ region of rat. The involvement of endogenous ATP in the development of TMJ inflammatory hyperalgesia was also determined by evaluating the effect of the general P2 receptor antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) on carrageenan-induced TMJ inflammatory hyperalgesia. Application of alpha,beta-meATP into the TMJ region of rats produced significant nociceptive responses that were significantly reduced by the co-application of lidocaine N-ethyl bromide quaternary salt, QX-314, (2%) or of the P2 receptor antagonist PPADS. Co-application of PPADS with carrageenan into the TMJ significantly reduced inflammatory hyperalgesia. The results indicate that functional P2X receptors are present in the TMJ and suggest that endogenous ATP may play a role in TMJ inflammatory pain mechanisms possibly by acting primarily in these receptors.     

Dural afferents express acid-sensing ion channels: a role for decreased meningeal pH in migraine headache

Migraine headache is one of the most common neurological disorders. The pathological conditions that directly initiate afferent pain signaling are poorly understood. In trigeminal neurons retrogradely labeled from the cranial meninges, we have recorded pH-evoked currents using whole-cell patch-clamp electrophysiology. Approximately 80% of dural-afferent neurons responded to a pH 6.0 application with a rapidly activating and rapidly desensitizing ASIC-like current that often exceeded 20 nA in amplitude. Inward currents were observed in response to a wide range of pH values and 30% of the neurons exhibited inward currents at pH 7.1. These currents led to action potentials in 53%, 30% and 7% of the dural afferents at pH 6.8, 6.9 and 7.0, respectively. Small decreases in extracellular pH were also able to generate sustained window currents and sustained membrane depolarizations. Amiloride, a non-specific blocker of ASIC channels, inhibited the peak currents evoked upon application of decreased pH while no inhibition was observed upon application of TRPV1 antagonists. The desensitization time constant of pH 6.0-evoked currents in the majority of dural afferents was less than 500 ms which is consistent with that reported for ASIC3 homomeric or heteromeric channels. Finally, application of pH 5.0 synthetic-interstitial fluid to the dura produced significant decreases in facial and hind-paw withdrawal threshold, an effect blocked by amiloride but not TRPV1 antagonists, suggesting that ASIC activation produces migraine-related behavior in vivo. These data provide a cellular mechanism by which decreased pH in the meninges following ischemic or inflammatory events directly excites afferent pain-sensing neurons potentially contributing to migraine headache.     

Complexities of measuring antagonist potency at P2X(7) receptor orthologs

The ability of P2 antagonists to affect agonist-stimulated fluorescent dye accumulation in cells expressing human, rat, or mouse P2X(7) receptors was examined. Several compounds, including pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), which was previously thought to be a weak P2X(7) receptor antagonist, possessed high potency (nanomolar IC(50)) at human and rat P2X(7) receptors. However, there were species differences in antagonist potency with PPADS, pyridoxal 5'-phosphate (P5P), and periodate-oxidized ATP (OxATP) exhibiting 20- to 500-fold higher potency for human than for mouse P2X(7) receptors. HMA (5-(N,N-hexamethylene)amiloride) was also selective for human over rat P2X(7) receptors but potentiated responses at mouse P2X(7) receptors. Coomassie Brilliant Blue G (CBB) was a nonselective antagonist with high potency at mouse P2X(7) receptors (IC(50) approximately 100 nM). All compounds were noncompetitive antagonists, and potency could only be quantified by measuring IC(50) values. These values were similar when determined against EC(50) concentrations of ATP or 2'- and 3'-O-4(-benzoylbenzoyl)-ATP and, for most compounds, only slightly (3- to 5-fold) affected by agonist concentration. However, IC(50) values for KN62 (1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine) and suramin, varied up to 25-fold depending upon agonist concentration. Furthermore, IC(50) values for KN62 and OxATP were 10-fold lower at 22 degrees C than at 37 degrees C, whereas IC(50) values for PPADS, P5P, suramin, and OxATP were up to 20-fold lower in NaCl than in sucrose buffer. Potency estimates for CBB and PPADS decreased 5-fold in the presence of bovine serum albumin, possibly due to protein binding. Given the species differences, and the effects of assay conditions on antagonist potency, caution must be exercised when interpreting results obtained with the available antagonists.     

Inhibition of transient receptor potential vanilloid‐1 confers neuroprotection,reduces tumor necrosis factor‐alpha,and increases IL‐10 in a rat stroke model

Stroke is a major cause of mortality and long‐term disability in adults. Transient receptor potential vanilloid‐1 (TRPV1) plays a crucial role in neuroinflammation. In this study, the effects of TRPV1 agonist (capsaicin) and antagonist (AMG9810) on cerebral ischemia were investigated. Forty male Wistar rats were assigned to the following experimental groups: sham, vehicle) ischemic), AMG9810 (selective TRPV1 antagonist, 0.5 mg/kg; 3 h after stroke), and capsaicin (1 mg/kg; 3 h after stroke). Stroke was induced by permanent middle cerebral artery occlusion and neurological deficits were evaluated 1, 3, and 7 days after stroke. Then, infarct volume, brain edema, body temperature, mRNA expression of TRPV1, and serum concentrations of tumor necrosis factor‐alpha (TNF‐α) and IL‐10 were measured. Compared to the vehicle group, AMG9810 significantly decreased the infarct volume (P < 0.01). Latency for the removal of sticky labels from the forepaw and the hanging time were significantly decreased and increased, respectively, following administration of AMG9810 (P < 0.01 and P < 0.001 vs. vehicle) 3 and 7 days after stroke. Compared to the sham group, the mRNA expression of TRPV1 was significantly increased in vehicle group (P < 0.01). Administration of AMG9810 significantly increased the anti‐inflammatory cytokine IL‐10 and decreased the inflammatory cytokine TNF‐α (P < 0.05). Moreover, our results indicate that AMG9810 might a promising candidate for the hypothermic treatment of stroke. The findings also suggest a key role for AMG9810 in reducing inflammation after stroke and imply that TRPV1 could be a potential target for the treatment of ischemic stroke.     

Chronic hyperalgesia induced by repeated acid injections in muscle is abolished by the loss of ASIC3, but not ASIC1

Clinically, chronic pain and hyperalgesia induced by muscle injury are disabling and difficult to treat. Cellular and molecular mechanisms underlying chronic muscle-induced hyperalgesia are not well understood. For this reason, we developed an animal model where repeated injections of acidic saline into one gastrocnemius muscle produce bilateral, long-lasting mechanical hypersensitivity of the paw (i.e. hyperalgesia) without associated tissue damage. Since acid sensing ion channels (ASICs) are found on primary afferent fibers and respond to decreases in pH, we tested the hypothesis that ASICs on primary afferent fibers innervating muscle are critical to development of hyperalgesia and central sensitization in response to repeated intramuscular acid. Dorsal root ganglion neurons innervating muscle express ASIC3 and respond to acidic pH with fast, transient inward and sustained currents that resemble those of ASICs. Mechanical hyperalgesia produced by repeated intramuscular acid injections is prevented by prior treatment of the muscle with the non-selective ASIC antagonist, amiloride, suggesting ASICs might be involved. ASIC3 knockouts do not develop mechanical hyperalgesia to repeated intramuscular acid injection when compared to wildtype littermates. In contrast, ASIC1 knockouts develop hyperalgesia similar to their wildtype littermates. Extracellular recordings of spinal wide dynamic range (WDR) neurons from wildtype mice show an expansion of the receptive field to include the contralateral paw, an increased response to von Frey filaments applied to the paw both ipsilaterally and contralaterally, and increased response to noxious pinch contralaterally after the second intramuscular acid injection. These changes in WDR neurons do not occur in ASIC3 knockouts. Thus, activation of ASIC3s on muscle afferents is required for development of mechanical hyperalgesia and central sensitization that normally occurs in response to repeated intramuscular acid. Therefore, interfering with ASIC3 might be of benefit in treatment or prevention of chronic hyperalgesia.     

Inhibition of acid-sensing ion channels reduces the hypothalamus–pituitary–adrenal axis activity and ameliorates depression-like behavior in rats

Depression is the leading cause of disability worldwide, and its treatment represents a major clinical challenge. The hypothalamus–pituitary–adrenal (HPA) axis has been known to play a crucial role in depression and serves as a target for antidepressants. Acid-sensing ion channels (ASICs) are widely expressed in the nervous system and may be implicated in depression. Whether ASICs could act on the HPA axis to affect depression-related behaviors is not fully understood. In this study, we investigated the effect of inhibition of ASICs on the HPA axis activity in chronic stress-subjected rats. We found that treatment with the ASIC selective antagonist amiloride reversed chronic stress-induced elevation of adrenocorticotropic hormone (ACTH) and corticosterone in serum, which is reflective of the HPA axis activity. In addition, amiloride also alleviated chronic stress-induced anhedonia-like behavior. These results suggest that inhibition of ASICs may act on the HPA axis to alleviate the symptoms of depression.

Depression is the leading cause of disability worldwide, and its treatment represents a major clinical challenge.     

Functional interactions between NMDA receptors and TRPV1 in trigeminal sensory neurons mediate mechanical hyperalgesia in the rat masseter muscle

The NMDA and TRPV1 receptors that are expressed in sensory neurons have been independently demonstrated to play important roles in peripheral pain mechanisms. In the present study, we investigated whether the 2 receptor-channel systems form a functional complex that provides the basis for the development of mechanical hyperalgesia. In the masseter muscle, direct application of NMDA induced a time-dependent increase in mechanical sensitivity, which was significantly blocked when the muscle was pretreated with a specific TRPV1 antagonist, AMG9810. The NR1 subunit of the NMDA receptor and TRPV1 were coexpressed in 32% of masseter afferents in trigeminal ganglia (TG). Furthermore, NR1 and NR2B formed protein-protein complexes with TRPV1 in TG as demonstrated by coimmunoprecipitation experiments. Calcium imaging analyses further corroborated that NMDA and TRPV1 receptors functionally interact. In TG culture, application of NMDA resulted in phosphorylation of serine, but not threonine or tyrosine, residues of TRPV1 in a time course similar to that of the development of NMDA-induced mechanical hyperalgesia. The NMDA-induced phosphorylation was significantly attenuated by CaMKII and PKC inhibitors, but not by a PKA inhibitor. Consistent with the biochemical data, the NMDA-induced mechanical hyperalgesia was also effectively blocked when the muscle was pretreated with a CaMKII or PKC inhibitor. Thus, NMDA receptors and TRPV1 functionally interact via CaMKII and PKC signaling cascades and contribute to mechanical hyperalgesia. These data offer novel mechanisms by which 2 ligand-gated channels in sensory neurons interact and reinforce the notion that TRPV1 functions as a signal integrator under pathological conditions.     

Involvement of ATP and its receptors on nociception in rat model of masseter muscle pain

The exact mechanism of the masseter muscle pain recognized as a prominent symptom in temporomandibular disorders remains unclear, although it is clinically known that excessive muscular contraction causes tenderness in masseter muscles. It has been demonstrated that P2X3 receptors (P2X3Rs) in sensory neurons play a role in pain signaling from the periphery. We determined the role of P2X(3)R on pressure pain and mechanical hyperalgesia in a newly developed rat model of masseter muscle pain. The pain in the masseter muscle was assessed by the pressure pain threshold (PPT), which was defined as the amount of pressure required to induce head flinching. In naive animals, systemic treatment with morphine was associated with increase of PPTs. Changes in PPTs were examined after administration of P2XR agonists or antagonists into the masseter muscle. The masseter muscle injection of alpha,beta-meATP (P2X(1,3,2/3)R-specific agonist) induced a significantly greater behavioral response than its vehicle. This enhanced response was completely blocked by the co-application of alpha,beta-meATP with PPADS (P2X(1,2,3,5,1/5,2/3)R-specific antagonist). Excessive contraction in masseter muscle was produced by electrical stimulation. The exerted masseter muscles showed a significant reduction in PPTs indicating the induction of mechanical hyperalgesia of the muscle. Moreover, administration of PPADS to the exerted masseter muscles produced a complete recovery of reducing PPT. Immunohistochemically, the number of P2X3R-positive neurons innervating the masseter muscles increased in the excessively contracted condition in trigeminal ganglia. Our results suggested that P2X3R plays an important role in pressure pain and mechanical hyperalgesia in masseter muscle caused by excessive muscular contraction.     

Structure and function of nociceptive neuronal receptors

It has been well known that pain is caused by nociceptive stimulation such as protons (i.e. acidic solutions), heat and capsaicin, a pungent ingredient of chilli peppers. For a long period, the signal transduction mechanism of pain activated by these nociceptive stimuli has not been clarified. Recent advance, especially the identification of TRPV1 receptor (for which capsaicin, protons and heat are ligands), P2X and P2Y receptor (for which ATP is a ligand) and acid sensing ion channel made a remarkable progress in understanding the mechanism of nociceptive neurons. This article reviews the structures and functions of nociceptive neuronal system particularly in TRPV1 receptor, P2X and P2Y receptors.     

Pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS), a putative P2Y(1) receptor antagonist, blocks signaling at a site distal to the receptor in Madin-Darby canine kidney-D(1) cells

Substantial evidence documents the potential importance of P2Y receptor subtypes in the regulation of cellular responses, but few selective antagonists exist for these receptors. In the current study, we assessed the use of pyridoxal-phosphate-6-azophenyl-2', 4'-disulfonate (PPADS) as a putative P2Y(1) receptor-selective blocker in Madin-Darby canine kidney (MDCK-D(1)) cells. We found that the key action of PPADS in MDCK-D(1) cells was blockade of signaling at a postreceptor site. PPADS blocked UTP (P2Y(2))-stimulated accumulation of cAMP [which is dependent on arachidonic acid (AA) metabolism by cyclooxygenase] but not that by 2-methyl thio-adenosine triphosphate (2MeSATP; which is independent of cyclooxygenase and has been attributed to P2Y(1) and P2Y(11) receptors). By contrast, PPADS inhibited AA release mediated by both 2MeSATP and UTP. PPADS displayed uncompetitive antagonism in blockade of AA release in response to 2MeSATP. PPADS also inhibited AA release stimulated by various nucleotides, phenylephrine, and bradykinin, implying that the effect does not involve the inhibition of a specific receptor. Because PPADS also inhibited ionomycin-, thapsigargin-, and phorbol-12-myristate-13-acetate-promoted AA release, it appears to act at a site distal to an increase in intracellular Ca(2+) transients or PKC activation. Inhibition of melittin-stimulated AA release by PPADS suggested that the target of PPADS action may either be a phospholipase A(2) (PLA(2)) or a site distal to PLA(2), but PPADS did not inhibit Ca(2+)-dependent PLA(2) activity in MDCK-D(1) cell homogenate. The data indicate that PPADS blocks AA release in response to multiple compounds and suggest caution in the use of this compound for distinguishing P2Y receptor subtypes.     

Mechanical allodynia and thermal hyperalgesia induced by experimental squamous cell carcinoma of the lower gingiva in rats.

We developed a rat model of oral cancer pain by inoculating cancer cells into the lower gingiva. A squamous cell carcinoma (SCC) derived from Fisher rats, SCC-158, was inoculated into the subperiosteal tissue on the lateral side of the lower gingiva in male Fisher rats. Inoculation of cancer cells induced marked mechanical allodynia and thermal hyperalgesia in the ipsilateral maxillary and mandibular nerve area. Infiltration of the tumor cells into the mandible and the completely encompassed inferior alveolar nerve was observed. Calcitonin gene-related peptide (CGRP)-, substance P (SP)-, ATP receptor (P2X(3))-, and capsaicin receptor (TRPV1)-immunoreactive cells strikingly increased in the small-cell group of trigeminal ganglia (TGs) after tumor cell inoculation. The TRPV1-immunoreactive cells also increased in the medium- and large-cell groups. Retrograde tracing combined with immunofluorescence techniques revealed the increased expression of peptides and the receptors in maxillary nerve afferent neurons. These results suggest that inoculation of SCC cells into the lower gingiva produces mechanical allodynia and thermal hyperalgesia, indicating the establishment of a novel rat model of oral cancer pain. Increased expression of CGRP, SP, P2X(3), and TRPV1 in the TG may be involved in the behavioral changes in this model. PERSPECTIVE: To clarify the mechanisms of oral cancer pain, we examined the expression of calcitonin gene-related peptide, substance P, ATP receptor P2X(3), and capsaicin receptor TRPV1 in trigeminal ganglia. Characterizations of these molecular systems which mediate pain perception are important to develop novel clinical tools for promoting relief of oral cancer pain.     

Pharmacological blockade of TRPV1 receptors modulates the effects of 6‐OHDA on motor and cognitive functions in a rat model of Parkinson's disease

TRPV1 receptors and cannabinoid system are considered as important modulators of basal ganglia functions, and their pharmacologic manipulation represents a promising therapy to alleviate Parkinson‐induced hypokinesia. Recent evidence suggests that the blockade of cannabinoid receptors might be beneficial to alleviate motor deficits observed in Parkinson's disease. In the present study, we have evaluated the effects of AMG₉₈₁₀, a selective antagonist of TRPV1 receptors, on the motor and cognitive functions in a rat model of Parkinson's disease generated by an intracerebroventricular injection of 6‐ hydroxydopamine (6‐OHDA) (200 μg per animal). The injection of 10 nmol of AMG₉₈₁₀ for a single dose (AMG1) and for 2 weeks (AMG14) partially attenuated the hypokinesia shown by these animals in motor function evaluation tests, whereas chronic administration of AMG had destructive effects on learning and memory in 6‐OHDA‐treated rats. Animals in the AMG 1 and AMG 14 groups showed an increased latency to fall in rotarod and grasping tests in each trials compared with 6‐OHDA‐treated rats (P < 0.01) and DMSO 1 and 14 groups (P < 0.05). Our data indicate that pharmacological blockade of TRPV1 receptors by AMG ₉₈₁₀ attenuates the hypokinetic effects of 6‐OHDA and that TRPV1 receptors play an important role in 6‐OHDA‐induced hypokinesia, athough elucidation of the neurochemical substrate involved in this process remains a major challenge for the future.