ASIC1 and ASIC3 Play Different Roles in the Development of Hyperalgesia After Inflammatory Muscle Injury

Acid-sensing ion channels (ASICs) respond to acidosis that normally occurs after inflammation. We examined the expression of ASIC1, ASIC2, and ASIC3 mRNAs in lumbar dorsal root ganglion neurons before and 24 hours after carrageenan-induced muscle inflammation. Muscle inflammation causes bilateral increases of ASIC2 and ASIC3 but not ASIC1 (neither ASIC1a nor ASIC1b) mRNA, suggesting differential regulation of ASIC1 versus ASIC2 and ASIC3 mRNA. Similar mRNA increases were observed after inflammation in knockout mice: ASIC2 mRNA increases in ASIC3-/- mice; ASIC2 and ASIC3 mRNAs increase in ASIC1-/- mice. Prior behavioral studies in ASIC3-/- mice showed deficits in secondary hyperalgesia (increased response to noxious stimuli outside the site of injury) but not primary hyperalgesia (increased response to noxious stimuli at the site of injury). In this study, we show that ASIC1-/- mice do not develop primary muscle hyperalgesia but develop secondary paw hyperalgesia. In contrast, and as expected, ASIC3-/- mice develop primary muscle hyperalgesia but do not develop secondary paw hyperalgesia. The pharmacological utility of the nonselective ASIC inhibitor A-317567, given locally, was tested. A-317567 reverses both the primary and the secondary hyperalgesia induced by carrageenan muscle inflammation. Thus, peripherally located ASIC1 and ASIC3 play different roles in the development of hyperalgesia after muscle inflammation.PerspectiveThis study shows changes in ASIC mRNA expression and behavioral hyperalgesia of C57Bl/6 (wild type), ASIC1-/-, and ASIC3-/- mice before and after the induction of muscle inflammation. A-317567 was effective in reversing hyperalgesia in these animals, suggesting the potential of ASICs as therapeutic targets for muscle inflammatory pain.     

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.     

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.     

ASIC3 in muscle mediates mechanical, but not heat, hyperalgesia associated with muscle inflammation

Peripheral initiators of muscle pain are virtually unknown, but likely key to development of chronic pain after muscle insult. The current study tested the hypothesis that ASIC3 in muscle is necessary for development of cutaneous mechanical, but not heat, hyperalgesia induced by muscle inflammation. Using mechanical and heat stimuli, we assessed behavioral responses in ASIC3-/- and ASIC3+/+ mice after induction of carrageenan muscle inflammation. ASIC3-/- mice did not develop cutaneous mechanical hyperalgesia after muscle inflammation when compared to ASIC3+/+ mice; heat hyperalgesia developed similarly between groups. We then tested if the phenotype could be rescued in ASIC3-/- mice by using a recombinant herpes virus vector to express ASIC3 in skin (where testing occurred) or muscle (where inflammation occurred). Infection of mouse DRG neurons with ASIC3-encoding virus resulted in functional expression of ASICs. Injection of ASIC3-encoding virus into muscle or skin of ASIC3-/- mice resulted in ASIC3 mRNA in DRG and protein expression in DRG and the peripheral injection site. Injection of ASIC3-encoding virus into muscle, but not skin, resulted in development of mechanical hyperalgesia similar to that observed in ASIC3+/+ mice. Thus, ASIC3 in primary afferent fibers innervating muscle is critical to development of hyperalgesia that results from muscle insult.     

Nociception and inflammatory hyperalgesia evaluated in rodents using infrared laser stimulation after Trpv1 gene knockout or resiniferatoxin lesion

TRPV1 is expressed in a subpopulation of myelinated Aδ and unmyelinated C-fibers. TRPV1⁺ fibers are essential for the transmission of nociceptive thermal stimuli and for the establishment and maintenance of inflammatory hyperalgesia. We have previously shown that high-power, short-duration pulses from an infrared diode laser are capable of predominantly activating cutaneous TRPV1⁺ Aδ-fibers. Here we show that stimulating either subtype of TRPV1⁺ fiber in the paw during carrageenan-induced inflammation or following hind-paw incision elicits pronounced hyperalgesic responses, including prolonged paw guarding. The ultrapotent TRPV1 agonist resiniferatoxin (RTX) dose-dependently deactivates TRPV1⁺ fibers and blocks thermal nociceptive responses in baseline or inflamed conditions. Injecting sufficient doses of RTX peripherally renders animals unresponsive to laser stimulation even at the point of acute thermal skin damage. In contrast, Trpv1−/− mice, which are generally unresponsive to noxious thermal stimuli at lower power settings, exhibit withdrawal responses and inflammation-induced sensitization using high-power, short duration Aδ stimuli. In rats, systemic morphine suppresses paw withdrawal, inflammatory guarding, and hyperalgesia in a dose-dependent fashion using the same Aδ stimuli. The qualitative intensity of Aδ responses, the leftward shift of the stimulus-response curve, the increased guarding behaviors during carrageenan inflammation or after incision, and the reduction of Aδ responses with morphine suggest multiple roles for TRPV1⁺ Aδ fibers in nociceptive processes and their modulation of pathological pain conditions.     

Selective targeting of ASIC3 using artificial miRNAs inhibits primary and secondary hyperalgesia after muscle inflammation

Acid-sensing ion channels (ASICs) are activated by acidic pH and may play a significant role in the development of hyperalgesia. Earlier studies show ASIC3 is important for induction of hyperalgesia after muscle insult using ASIC3−/− mice. ASIC3−/− mice lack ASIC3 throughout the body, and the distribution and composition of ASICs could be different from wild-type mice. We therefore tested whether knockdown of ASIC3 in primary afferents innervating muscle of adult wild-type mice prevented development of hyperalgesia to muscle inflammation. We cloned and characterized artificial miRNAs (miR-ASIC3) directed against mouse ASIC3 (mASIC3) to downregulate ASIC3 expression in vitro and in vivo. In CHO-K1 cells transfected with mASIC3 cDNA in culture, the miR-ASIC3 constructs inhibited protein expression of mASIC3 and acidic pH-evoked currents and had no effect on protein expression or acidic pH-evoked currents of ASIC1a. When miR-ASIC3 was used in vivo, delivered into the muscle of mice using a herpes simplex viral vector, both muscle and paw mechanical hyperalgesia were reduced after carrageenan-induced muscle inflammation. ASIC3 mRNA in DRG and protein levels in muscle were decreased in vivo by miR-ASIC3. In CHO-K1 cells co-transfected with ASIC1a and ASIC3, miR-ASIC3 reduced the amplitude of acidic pH-evoked currents, suggesting an overall inhibition in the surface expression of heteromeric ASIC3-containing channels. Our results show, for the first time, that reducing ASIC3 in vivo in primary afferent fibers innervating muscle prevents the development of inflammatory hyperalgesia in wild-type mice, and thus, may have applications in the treatment of musculoskeletal pain in humans.     

Peripheral Acid-Sensing Ion Channels and P2X Receptors Contribute to Mechanical Allodynia in a Rodent Thrombus-Induced Ischemic Pain Model

We have previously established a thrombus-induced ischemic pain (TIIP) model in the rat, which mimics the pathophysiology of ischemic pain in patients with peripheral arterial disease. Because ischemia commonly induces acidosis and ATP release, one of the goals of this study was to investigate the role of acid-sensing ion channels (ASICs), transient receptor potential vanilloid-1 (TRPV1) receptors, and P2X receptors in the maintenance of ischemia-induced mechanical allodynia (MA). To test this, amiloride (an ASIC blocker), AMG-9810 (a TRPV1 blocker), or PPADS (a P2Xs antagonist) was intraplantarly injected at day 3 after FeCl₂ application onto the femoral artery. Ipsilateral administration of amiloride or PPADS but not AMG-9810 dose-dependently reduced MA. However, contralateral amiloride or PPADS did not suppress contralateral MA. Interestingly, co-administration of submaximal doses of amiloride and PPADS produced a significantly prolonged suppression of MA. Furthermore, ipsilateral EGTA (a calcium chelator) or chelerythrine (a protein kinase C inhibitor) also significantly reduced MA. Collectively, these findings suggest that peripheral ASICs and P2X receptors are involved in the maintenance of TIIP, which is possibly mediated by a Ca²⁺–protein kinase C signaling mechanism. These results provide mechanistic information about peripheral ischemic nociception that may be useful for developing better therapeutic management of ischemic pain in patients with peripheral arterial disease.PerspectiveThe results of the current study demonstrate that peripheral administration of an ASICs blocker or P2X antagonist significantly suppress TIIP. Co-administration of submaximal doses of ASIC and P2X antagonists produced an even greater effect. These results implicate peripheral ASICs and P2X receptors in the maintenance of thrombus-induced ischemic pain.     

<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.     

TRPV1 is important for mechanical and heat sensitivity in uninjured animals and development of heat hypersensitivity after muscle inflammation

Inflammatory thermal hyperalgesia is principally mediated through transient receptor potential vanilloid 1 (TRPV1) channels, as demonstrated by prior studies using models of cutaneous inflammation. Muscle pain is significantly different from cutaneous pain, and the involvement of TRPV1 in hyperalgesia induced by muscle inflammation is unknown. We tested whether TRPV1 contributes to the development of mechanical and heat hypersensitivity of the paw in TRPV1(-/-) mice after muscle inflammation. Because TRPV1(-/-) mice lack TRPV1 at the site of inflammation (muscle) and at the testing site (paw), we do not know whether TRPV1 is important as a mediator of nociceptor sensitization in the muscle or as a heat sensor in the paw. Using recombinant herpesviruses, we reexpressed TRPV1 in TRPV1(-/-) mice in primary afferents innervating skin, muscle, or both to determine which sites were important for the behavioral deficits. Responses to repeated application of noxious mechanical stimuli to the hind paw were enhanced in TRPV1(-/-) mice; this was restored by reexpression of TRPV1 into skin. Withdrawal latencies to noxious heat were increased in TRPV1(-/-) mice; normal latencies were restored by reexpression of TRPV1 in both skin and muscle. Heat hypersensitivity induced by muscle inflammation did not develop in TRPV1(-/-) mice; mechanical hypersensitivity was similar between TRPV1(-/-) and TRPV1(+/+) mice. Heat hypersensitivity induced by muscle inflammation was restored by reexpression of TRPV1 into both muscle and skin of TRPV1(-/-) mice. These results suggest that TRPV1 serves as both a mediator of nociceptor sensitization at the site of inflammation and as a heat sensor at the paw.     

TRPV4 is necessary for trigeminal irritant pain and functions as a cellular formalin receptor

Detection of external irritants by head nociceptor neurons has deep evolutionary roots. Irritant-induced aversive behavior is a popular pain model in laboratory animals. It is used widely in the formalin model, where formaldehyde is injected into the rodent paw, eliciting quantifiable nocifensive behavior that has a direct, tissue-injury-evoked phase, and a subsequent tonic phase caused by neural maladaptation. The formalin model has elucidated many antipain compounds and pain-modulating signaling pathways. We have adopted this model to trigeminally innervated territories in mice. In addition, we examined the involvement of TRPV4 channels in formalin-evoked trigeminal pain behavior because TRPV4 is abundantly expressed in trigeminal ganglion (TG) sensory neurons, and because we have recently defined TRPV4’s role in response to airborne irritants and in a model for temporomandibular joint pain. We found TRPV4 to be important for trigeminal nocifensive behavior evoked by formalin whisker pad injections. This conclusion is supported by studies with Trpv4^−/− mice and TRPV4-specific antagonists. Our results imply TRPV4 in MEK-ERK activation in TG sensory neurons. Furthermore, cellular studies in primary TG neurons and in heterologous TRPV4-expressing cells suggest that TRPV4 can be activated directly by formalin to gate Ca²⁺. Using TRPA1-blocker and Trpa1^−/− mice, we found that both TRP channels co-contribute to the formalin trigeminal pain response. These results imply TRPV4 as an important signaling molecule in irritation-evoked trigeminal pain. TRPV4-antagonistic therapies can therefore be envisioned as novel analgesics, possibly for specific targeting of trigeminal pain disorders, such as migraine, headaches, temporomandibular joint, facial, and dental pain, and irritation of trigeminally innervated surface epithelia.     

Involvement of ASIC3 and Substance P in Therapeutic Ultrasound–Mediated Analgesia in Mouse Models of Fibromyalgia

Therapeutic ultrasound (tUS) is widely used in chronic muscle pain control. However, its analgesic molecular mechanism is still not known. Our objective is to reveal the mechanism of the tUS-induced analgesia in mouse models of fibromyalgia. We applied tUS in mice that have developed chronic hyperalgesia induced by intramuscular acidification and determined the tUS frequency at 3 MHz, dosage at 1 W/cm² (measured output as 6.3 mW/cm²) and 100% duty cycle for 3 minutes having the best analgesic effect. Pharmacological and genetic approaches were used to probe the molecular determinants involved in tUS-mediated analgesia. A second mouse model of fibromyalgia induced by intermittent cold stress was further used to validate the mechanism underlying the tUS-mediated analgesia. The tUS-mediated analgesia was abolished by a pretreatment of NK1 receptor antagonist—RP-67580 or knockout of substance P (Tac1^-/-). Besides, the tUS-mediated analgesia was abolished by ASIC3-selective antagonist APETx2 but not TRPV1-selective antagonist capsazepine, suggesting a role for ASIC3. Moreover, the tUS-mediated analgesia was attenuated by ASIC3-selective nonsteroid anti-inflammation drugs (NSAIDs)—aspirin and diclofenac but not by ASIC1a-selective ibuprofen. We next validated the antinociceptive role of substance P signaling in the model induced by intermittent cold stress, in which tUS-mediated analgesia was abolished in mice lacking substance P, NK1R, Asic1a, Asic2b, or Asic3 gene. tUS treatment could activate ASIC3-containing channels in muscle afferents to release substance P intramuscularly and exert an analgesic effect in mouse models of fibromyalgia. NSAIDs should be cautiously used or avoided in the tUS treatment.PerspectiveTherapeutic ultrasound showed analgesic effects against chronic mechanical hyperalgesia in the mouse model of fibromyalgia through the signaling pathways involving substance P and ASIC3-containing ion channels in muscle afferents. NSAIDs should be cautiously used during tUS treatment.     

TRPV1 Receptor in Expression of Opioid-Induced Hyperalgesia

Opiates are currently the mainstay for treatment of moderate to severe pain. However, prolonged administration of opiates has been reported to elicit hyperalgesia in animals, and examples of opiate-induced hyperalgesia have been reported in humans as well. Despite the potential clinical significance of such opiate-induced actions, the mechanisms of opiate-induced hypersensitivity remain unknown. The transient receptor potential vanilloid1 (TRPV1) receptor, a molecular sensor of noxious heat, acts as an integrator of multiple forms of noxious stimuli and plays an important role in the development of inflammation-induced hyperalgesia. Because animals treated with opiates show thermal hyperalgesia, we examined the possible role of TRPV1 receptors in the development of morphine-induced hyperalgesia using TRPV1 wild-type (WT) and knock-out (KO) mice and with administration of a TRPV1 antagonist in mice and rats. Administration of morphine by subcutaneous implantation of morphine pellets elicited both thermal and tactile hypersensitivity in TRPV1 WT mice but not in TRPV1 KO mice. Moreover, oral administration of a TRPV1 antagonist reversed both thermal and tactile hypersensitivity induced by sustained morphine administration in mice and rats. Immunohistochemical analyses indicate that sustained morphine administration modestly increases TRPV1 labeling in the dorsal root ganglia. In addition, sustained morphine increased flinching and plasma extravasation after peripheral stimulation with capsaicin, suggesting an increase in TRPV1 receptor function in the periphery in morphine-treated animals. Collectively, our data indicate that the TRPV1 receptor is an essential peripheral mechanism in expression of morphine-induced hyperalgesia.PerspectiveOpioid-induced hyperalgesia possibly limits the usefulness of opioids, emphasizing the value of alternative methods of pain control. We demonstrate that TRPV1 channels play an important role in peripheral mechanisms of opioid-induced hyperalgesia. Such information may lead to the discovery of analgesics lacking such adaptations and improving treatment of chronic pain.     

Isopentenyl pyrophosphate is a novel antinociceptive substance that inhibits TRPV3 and TRPA1 ion channels

Transient receptor potential ion channels (TRPs) expressed in the periphery sense and electrically transduce noxious stimuli to transmit the signals to the brain. Many natural and synthetic ligands for the sensory TRPs have been found, but little is known about endogenous inhibitors of these TRP channels. Recently, we reported that farnesyl pyrophosphate, an endogenous substance produced in the mevalonate pathway, is a specific activator for TRPV3. Here, we show that isopentenyl pyrophosphate (IPP), an upstream metabolite in the same pathway, is a dual inhibitor for TRPA1 and TRPV3. By using Ca²⁺ imaging and voltage clamp experiments with human embryo kidney cell heterologous expression system, cultured sensory neurons, and epidermal keratinocytes, we demonstrate that micromolar IPP suppressed responses to specific agonists of TRPA1 and TRPV3. Consistently, peripheral IPP administration attenuated TRPA1 and TRPV3 agonist-specific acute pain behaviors. Furthermore, local IPP pretreatment significantly reversed mechanical and thermal hypersensitivity of inflamed animals. Taken together, the present study suggests that IPP is a novel endogenous TRPA1 and TRPV3 inhibitor that causes local antinociception. Our results may provide useful chemical information to elucidate TRP physiology in peripheral pain sensation.     

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.     

Differential contribution of TRPV1 to thermal responses and tissue injury-induced sensitization of dorsal horn neurons in laminae I and V in the mouse

Our previous recordings from dorsal root ganglion and spinal lamina V neurons from TRPV1-mutant mice showed dramatic decreases in responses to temperatures near the activation threshold of this channel (43-49 degrees C). Somewhat unexpectedly, we only observed behavioral deficits in these mice at higher temperatures (50-58 degrees C). In the present study, we tested the hypothesis that the noxious heat-evoked pain behavior that persists in TRPV1-mutant mice reflects residual responsiveness of neurons in the superficial, but not deep, dorsal horn. To this end, we performed in vivo extracellular recordings of spinal nociresponsive neurons in laminae I and V in wild type (WT) and TRPV1 mutant mice. Neurons in WT and mutant mice from both laminae did not differ in their spontaneous activity or evoked responses to mechanical or cold stimuli. By contrast, most lamina I neurons from mutant mice responded to noxious heat with significantly higher thresholds than in WT mice. In contrast, lamina V neurons from mutant mice were virtually unresponsive to noxious heat before and after topical mustard oil-induced tissue injury. Interestingly, lamina I neurons in mutant mice displayed thermal sensitization following tissue injury, comparable in magnitude, but of shorter duration, than in WT mice. We conclude that TRPV1 is necessary for noxious heat-evoked responses of lamina V neurons, both before and after tissue injury. It is also an essential contributor to the normal activation threshold of lamina I neurons to noxious heat and for the full duration of thermal sensitization of lamina I neurons following injury. Finally, our results suggest that the processing of noxious thermal messages by neurons in lamina I involves convergent inputs from a heterogeneous population of primary afferent thermal nociceptors.     

TRPV1 channels make major contributions to behavioral hypersensitivity and spontaneous activity in nociceptors after spinal cord injury

Chronic neuropathic pain is often a severe and inadequately treated consequence of spinal cord injury (SCI). Recent findings suggest that SCI pain is promoted by spontaneous activity (SA) generated chronically in cell bodies of primary nociceptors in dorsal root ganglia (DRG). Many nociceptors express transient receptor potential V1 (TRPV1) channels, and in a preceding study most dissociated DRG neurons exhibiting SA were excited by the TRPV1 activator, capsaicin. The present study investigated roles of TRPV1 channels in behavioral hypersensitivity and nociceptor SA after SCI. Contusive SCI at thoracic segment T10 increased expression of TRPV1 protein in lumbar DRG 1 month after injury and enhanced capsaicin-evoked ion currents and Ca²⁺ responses in dissociated small DRG neurons. A major role for TRPV1 channels in pain-related behavior was indicated by the ability of a specific TRPV1 antagonist, AMG9810, to reverse SCI-induced hypersensitivity of hind limb withdrawal responses to mechanical and thermal stimuli at a dose that did not block detection of noxious heat. Similar reversal of behavioral hypersensitivity was induced by intrathecal oligodeoxynucleotides antisense to TRPV1, which knocked down TRPV1 protein and reduced capsaicin-evoked currents. TRPV1 knockdown also decreased the incidence of SA in dissociated nociceptors after SCI. Prolonged application of very low concentrations of capsaicin produced nondesensitizing firing similar to SA, and this effect was enhanced by prior SCI. These results show that TRPV1 makes important contributions to pain-related hypersensitivity long after SCI, and suggest a role for TRPV1-dependent enhancement of nociceptor SA that offers a promising target for treating chronic pain after SCI.     

Lack of TRPV1 inhibits cystitis-induced increased mechanical sensitivity in mice

Transient receptor potential vanilloid 1 (TRPV1) is highly expressed in primary afferent neurons. Tissue damage generates an array of chemical mediators that activate and sensitize afferent nerve fibers, and sensitization of afferent nerve fibers plays an important role in development of visceral pain. We investigated participation of TRPV1 in visceral pain associated with bladder inflammation induced in mice by systemic treatment with cyclophosphamide or intravesical instillation of acrolein. The effects of experimental cystitis on bladder function (an indicator of visceral pain) and the threshold of response to mechanical or thermal stimuli of the hind paws were investigated using TRPV1 knock-out (KO) and congenic wild-type (WT) mice. We found that cystitis induced bladder mechanical hyperreactivity and increased mechanical sensitivity of hind paws in WT, but not in TRPV1 KO mice. Lack of functional TRPV1 did not inhibit development of histological evidence of bladder inflammation, or increased expression of mRNAs for nerve growth factor, endothelial nitric oxide synthase, cyclooxygenase-2 and bradykinin receptors in urothelium. Cystitis did not affect the threshold of response to thermal stimuli in WT or KO mice. These results suggest that TRPV1 is essential for cystitis-induced bladder mechanical hyperreactivity. Also, TRPV1 participates in development of visceral pain, as reflected by referred increased mechanosensitivity in peripheral tissues in the presence of visceral inflammation.     

TRPM8, but not TRPA1, is required for neural and behavioral responses to acute noxious cold temperatures and cold-mimetics in vivo

Somatosensory neurons detect environmental stimuli, converting external cues into neural activity that is relayed first to second-order neurons in the spinal cord. The detection of cold is proposed to be mediated by the ion channels TRPM8 and TRPA1. However, there is significant debate regarding the role of each channel in cold-evoked pain, complicating their potential as drug targets for conditions such as cold allodynia and hyperalgesia. To address this debate, we generated mice lacking functional copies of both channels and examined behaviors and neural activity in response to painful cold and noxious cooling compounds. Whereas normal mice display a robust preference for warmth over cold, both TRPM8-null (TRPM8^−/−) and TRPM8/TRPA1 double-knockout mice (DKO) display no preference until temperatures reach the extreme noxious range. Additionally, in contrast to wildtype mice that avoid touching cold surfaces, mice lacking TRPM8 channels display no such avoidance and explore noxious cold surfaces, even at 5 °C. Furthermore, nocifensive behaviors to the cold-mimetic icilin are absent in TRPM8^−/− and DKO mice, but are retained in TRPA1-nulls (TRPA1^−/−). Finally, neural activity, measured by expression of the immediate-early gene c-fos, evoked by hindpaw stimulation with noxious cold, menthol, or icilin is reduced in TRPM8^−/− and DKO mice, but not in TRPA1^−/− animals. Thus our results show that noxious cold signaling is exclusive to TRPM8, mediating neural and behavioral responses to cold and cold-mimetics, and that TRPA1 is not required for acute cold pain in mammals.     

Medicinal Chemistry,Pharmacology, and Clinical Implications of TRPV1 Receptor Antagonists

Transient receptor potential vanilloid 1 (TRPV1) is an ion channel expressed on sensory neurons triggering an influx of cations. TRPV1 receptors function as homotetramers responsive to heat, proinflammatory substances, lipoxygenase products, resiniferatoxin, endocannabinoids, protons, and peptide toxins. Its phosphorylation increases sensitivity to both chemical and thermal stimuli, while desensitization involves a calcium‐dependent mechanism resulting in receptor dephosphorylation. TRPV1 functions as a sensor of noxious stimuli and may represent a target to avoid pain and injury. TRPV1 activation has been associated to chronic inflammatory pain and peripheral neuropathy. Its expression is also detected in nonneuronal areas such as bladder, lungs, and cochlea where TRPV1 activation is responsible for pathology development of cystitis, asthma, and hearing loss. This review offers a comprehensive overview about TRPV1 receptor in the pathophysiology of chronic pain, epilepsy, cough, bladder disorders, diabetes, obesity, and hearing loss, highlighting how drug development targeting this channel could have a clinical therapeutic potential. Furthermore, it summarizes the advances of medicinal chemistry research leading to the identification of highly selective TRPV1 antagonists and their analysis of structure–activity relationships (SARs) focusing on new strategies to target this channel.     

Antinociceptive pharmacology of N-(4-chlorobenzyl)-N'-(4-hydroxy-3-iodo-5-methoxybenzyl) thiourea, a high-affinity competitive antagonist of the transient receptor potential vanilloid 1 receptor

The transient receptor potential vanilloid 1 receptor (TRPV1) is expressed predominantly in a subset of primary afferent nociceptors. Due to its specific anatomical location and its pivotal role as a molecular integrator for noxious thermal and chemical stimuli, there is considerable interest to develop TRPV1 antagonists for the treatment of pain. Recently, N-(4-chlorobenzyl)-N'-(4-hydroxy-3-iodo-5-methoxybenzyl) thiourea (IBTU) was synthesized, and it was found in vitro to be a high-affinity competitive antagonist of cytoplasmic, but not intracellular, TRPV1. In this study, we examined the in vivo antinociceptive activity of IBTU in several acute and inflammatory pain models in mice. Our emphasis was on nociceptive pathways that are likely mediated by TRPV1, including capsaicin-, noxious heat-, and proton (including inflammation)-induced nociception tests. Capsazepine was used as a positive control in these experiments. IBTU dose-dependently blocked the capsaicin-induced nociception, confirming its antagonism at TRPV1 in vivo. By itself, IBTU produced significant antinociception, because it significantly prolonged the tail-flick latency in a dose-dependent manner. IBTU also blocked both early and late phases of the formalin-induced flinching response as well as acetic acid-induced writhing behavior. Moreover, IBTU inhibited the complete Freund's adjuvant-induced persistent hyperalgesia. Taken together, these data demonstrate that IBTU acts as a TRPV1 antagonist in vivo, and they suggest that it may be of therapeutic use for the treatment of pain.