Oxidative stress mediates thalidomide-induced pain by targeting peripheral TRPA1 and central TRPV4.

BACKGROUND: The mechanism underlying the pain symptoms associated with chemotherapeutic-induced peripheral neuropathy (CIPN) is poorly understood. Transient receptor potential ankyrin 1 (TRPA1), TRP vanilloid 4 (TRPV4), TRPV1, and oxidative stress have been implicated in several rodent models of CIPN-evoked allodynia. Thalidomide causes a painful CIPN in patients via an unknown mechanism. Surprisingly, the pathway responsible for such proalgesic response has not yet been investigated in animal models. RESULTS: Here, we reveal that a single systemic administration of thalidomide and its derivatives, lenalidomide and pomalidomide, elicits prolonged (~ 35 days) mechanical and cold hypersensitivity in C57BL/6J mouse hind paw. Pharmacological antagonism or genetic deletion studies indicated that both TRPA1 and TRPV4, but not TRPV1, contribute to mechanical allodynia, whereas cold hypersensitivity was entirely due to TRPA1. Thalidomide per se did not stimulate recombinant and constitutive TRPA1 and TRPV4 channels in vitro, which, however, were activated by the oxidative stress byproduct, hydrogen peroxide. Systemic treatment with an antioxidant attenuated mechanical and cold hypersensitivity, and the increase in oxidative stress in hind paw, sciatic nerve, and lumbar spinal cord produced by thalidomide. Notably, central (intrathecal) or peripheral (intraplantar) treatments with channel antagonists or an antioxidant revealed that oxidative stress-dependent activation of peripheral TRPA1 mediates cold allodynia and part of mechanical allodynia. However, oxidative stress-induced activation of central TRPV4 mediated the residual TRPA1-resistant component of mechanical allodynia. CONCLUSIONS: Targeting of peripheral TRPA1 and central TRPV4 may be required to attenuate pain associated with CIPN elicited by thalidomide and related drugs.

Transient receptor potential ankyrin 1 contributes to somatic pain hypersensitivity in experimental colitis.

Pain evoked by visceral inflammation is often 'referred' to the somatic level. Transient receptor potential ankyrin 1 (TRPA1) has been reported to contribute to visceral pain-like behavior in dextran sulfate sodium (DSS)-evoked colitis. However, the role of TRPA1 in somatic component of hypersensitivity due to visceral inflammation is unknown. The present study investigated the role of TRPA1 in colitis-evoked mechanical hypersensitivity at the somatic level. Colitis was induced in mice by adding DSS to drinking water for one week. Control and DSS-treated mice were tested for various parameters of colitis as well as mechanical pain sensitivity in abdominal and facial regions. DSS treatment caused mechanical hypersensitivity in the abdominal and facial skin. Pharmacological blockade or genetic deletion of TRPA1 prevented the colitis-associated mechanical hypersensitivity in the abdominal and facial skin areas although the severity of colitis remained unaltered. DSS treatment increased expression of TRPA1 mRNA in cultured dorsal root ganglion (DRG) neurons, but not trigeminal ganglion neurons, and selectively enhanced currents evoked by the TRPA1 agonist, allyl isothiocyanate, in cultured DRG neurons. Our findings indicate that the TRPA1 channel contributes to colitis-associated mechanical hypersensitivity in somatic tissues, an effect associated with upregulation of TRPA1 expression and responsiveness in DRG nociceptors.

Dacarbazine alone or associated with melanoma-bearing cancer pain model induces painful hypersensitivity by TRPA1 activation in mice.

Antineoplastic therapy has been associated with pain syndrome development characterized by acute and chronic pain. The chemotherapeutic agent dacarbazine, used mainly to treat metastatic melanoma, is reported to cause painful symptoms, compromising patient quality of life. Evidence has proposed that transient receptor potential ankyrin 1 (TRPA1) plays a critical role in chemotherapy-induced pain syndrome. Here, we investigated whether dacarbazine causes painful hypersensitivity in naive or melanoma-bearing mice and the involvement of TRPA1 in these models. Mouse dorsal root ganglion (DRG) neurons and human TRPA1-transfected HEK293 (hTRPA1-HEK293) cells were used to evaluate the TRPA1-mediated calcium response evoked by dacarbazine. Mechanical and cold allodynia were evaluated after acute or repeated dacarbazine administration in naive mice or after inoculation of B16-F10 melanoma cells in C57BL/6 mice. TRPA1 involvement was investigated by using pharmacological and genetic tools (selective antagonist or antisense oligonucleotide treatment and Trpa1 knockout mice). Dacarbazine directly activated TRPA1 in hTRPA1-HEK293 cells and mouse DRG neurons and appears to sensitize TRPA1 indirectly by generating oxidative stress products. Moreover, dacarbazine caused mechanical and cold allodynia in naive but not Trpa1 knockout mice. Also, dacarbazine-induced nociception was reduced by the pharmacological TRPA1 blockade (antagonism), antioxidants, and by ablation of TRPA1 expression. TRPA1 pharmacological blockade also reduced dacarbazine-induced nociception in a tumor-associated pain model. Thus, dacarbazine causes nociception by TRPA1 activation, indicating that this receptor may represent a pharmacological target for treating chemotherapy-induced pain syndrome in cancer patients submitted to antineoplastic treatment with dacarbazine.

Schwann cells expressing nociceptive channel TRPA1 orchestrate ethanol-evoked neuropathic pain in mice.

Excessive alcohol consumption is associated with spontaneous burning pain, hyperalgesia, and allodynia. Although acetaldehyde has been implicated in the painful alcoholic neuropathy, the mechanism by which the ethanol metabolite causes pain symptoms is unknown. Acute ethanol ingestion caused delayed mechanical allodynia in mice. Inhibition of alcohol dehydrogenase (ADH) or deletion of transient receptor potential ankyrin 1 (TRPA1), a sensor for oxidative and carbonyl stress, prevented allodynia. Acetaldehyde generated by ADH in both liver and Schwann cells surrounding nociceptors was required for TRPA1-induced mechanical allodynia. Plp1-Cre Trpa1fl/fl mice with a tamoxifen-inducible specific deletion of TRPA1 in Schwann cells revealed that channel activation by acetaldehyde in these cells initiates a NADPH oxidase-1-dependent (NOX1-dependent) production of hydrogen peroxide (H2O2) and 4-hydroxynonenal (4-HNE), which sustains allodynia by paracrine targeting of nociceptor TRPA1. Chronic ethanol ingestion caused prolonged mechanical allodynia and loss of intraepidermal small nerve fibers in WT mice. While Trpa1-/- or Plp1-Cre Trpa1fl/fl mice did not develop mechanical allodynia, they did not show any protection from the small-fiber neuropathy. Human Schwann cells express ADH/TRPA1/NOX1 and recapitulate the proalgesic functions of mouse Schwann cells. TRPA1 antagonists might attenuate some symptoms of alcohol-related pain.

Migraine-provoking substances evoke periorbital allodynia in mice.

BACKGROUND: Administration of endogenous mediators or exogenous chemicals in migraine patients provoke early headaches and delayed migraine-like attacks. Although migraine provoking substances are normally vasodilators, dilation of arterial vessels does not seem to be the sole contributing factor, and the underlying mechanisms of the delayed migraine pain are mostly unknown. Sustained mechanical allodynia is a common response associated with the local administration of various proalgesic substances in experimental animals and humans. Here, we investigated the ability of a series of endogenous mediators which provoke or do not provoke migraine in patients, to cause or not cause mechanical allodynia upon their injection in the mouse periorbital area. METHODS: Mechanical allodynia was assessed with the von Frey filament assay. Stimuli were given by subcutaneous injection in the periorbital area of C57BL/6J mice; antagonists were administered by local and systemic injections. RESULTS: Calcitonin gene related peptide (CGRP), but not adrenomedullin and amylin, pituitary adenylyl cyclase activating peptide (PACAP), but not vasoactive intestinal polypeptide (VIP), histamine, prostaglandin E2 (PGE2) and prostacyclin (PGI2), but not PGF2α, evoked a dose-dependent periorbital mechanical allodynia. The painful responses were attenuated by systemic or local (periorbital) administration of antagonists for CGRP (CLR/RAMP1), PACAP (PAC-1), histamine H1, PGE2 (EP4), and PGI2 (IP) receptors, respectively. CONCLUSIONS: The correspondence between substances that provoke (CGRP; PACAP, histamine, PGE2, PGI2), or do not provoke (VIP and PGF2α), migraine-like attacks in patients and periorbital allodynia in mice suggests that the study of allodynia in mice may provide information on the proalgesic mechanisms of migraine-provoking agents in humans. Results underline the ability of migraine-provoking substances to initiate mechanical allodynia by acting on peripheral terminals of trigeminal afferents.

The acyl-glucuronide metabolite of ibuprofen has analgesic and anti-inflammatory effects via the TRPA1 channel.

Ibuprofen is a widely used non-steroidal anti-inflammatory drug (NSAID) that exerts analgesic and anti-inflammatory actions. The transient receptor potential ankyrin 1 (TRPA1) channel, expressed primarily in nociceptors, mediates the action of proalgesic and inflammatory agents. Ibuprofen metabolism yields the reactive compound, ibuprofen-acyl glucuronide, which, like other TRPA1 ligands, covalently interacts with macromolecules. To explore whether ibuprofen-acyl glucuronide contributes to the ibuprofen analgesic and anti-inflammatory actions by targeting TRPA1, we used in vitro tools (TRPA1-expressing human and rodent cells) and in vivo mouse models of inflammatory pain. Ibuprofen-acyl glucuronide, but not ibuprofen, inhibited calcium responses evoked by reactive TRPA1 agonists, including allyl isothiocyanate (AITC), in cells expressing the recombinant and native human channel and in cultured rat primary sensory neurons. Responses by the non-reactive agonist, menthol, in a mutant human TRPA1 lacking key cysteine-lysine residues, were not affected. In addition, molecular modeling studies evaluating the covalent interaction of ibuprofen-acyl glucuronide with TRPA1 suggested the key cysteine residue C621 as a probable alkylation site for the ligand. Local administration of ibuprofen-acyl glucuronide, but not ibuprofen, in the mouse hind paw attenuated nociception by AITC and other TRPA1 agonists and the early nociceptive response (phase I) to formalin. Systemic ibuprofen-acyl glucuronide and ibuprofen, but not indomethacin, reduced phase I of the formalin response. Carrageenan-evoked allodynia in mice was reduced by local ibuprofen-acyl glucuronide, but not by ibuprofen, whereas both drugs attenuated PGE2 levels. Ibuprofen-acyl glucuronide, but not ibuprofen, inhibited the release of IL-8 evoked by AITC from cultured bronchial epithelial cells. The reactive ibuprofen metabolite selectively antagonizes TRPA1, suggesting that this novel action of ibuprofen-acyl glucuronide might contribute to the analgesic and anti-inflammatory activities of the parent drug.

TRPA1 mediates the antinociceptive properties of the constituent of Crocus sativus L., safranal.

Safranal, contained in Crocus sativus L., exerts anti-inflammatory and analgesic effects. However, the underlying mechanisms for such effects are poorly understood. We explored whether safranal targets the transient receptor potential ankyrin 1 (TRPA1) channel, which in nociceptors mediates pain signals. Safranal by binding to specific cysteine/lysine residues, stimulates TRPA1, but not the TRP vanilloid 1 and 4 channels (TRPV1 and TRPV4), evoking calcium responses and currents in human cells and rat and mouse dorsal root ganglion (DRG) neurons. Genetic deletion or pharmacological blockade of TRPA1 attenuated safranal-evoked release of calcitonin gene-related peptide (CGRP) from rat and mouse dorsal spinal cord, and acute nociception in mice. Safranal contracted rat urinary bladder isolated strips in a TRPA1-dependent manner, behaving as a partial agonist. After exposure to safranal the ability of allyl isothiocyanate (TRPA1 agonist), but not that of capsaicin (TRPV1 agonist) or GSK1016790A (TRPV4 agonist), to evoke currents in DRG neurons, contraction of urinary bladder strips and CGRP release from spinal cord slices in rats, and acute nociception in mice underwent desensitization. As previously shown for other herbal extracts, including petasites or parthenolide, safranal might exert analgesic properties by partial agonism and selective desensitization of the TRPA1 channel.

TRPA1/NOX in the soma of trigeminal ganglion neurons mediates migraine-related pain of glyceryl trinitrate in mice.

Glyceryl trinitrate is administered as a provocative test for migraine pain. Glyceryl trinitrate causes prolonged mechanical allodynia in rodents, which temporally correlates with delayed glyceryl trinitrate-evoked migraine attacks in patients. However, the underlying mechanism of the allodynia evoked by glyceryl trinitrate is unknown. The proalgesic transient receptor potential ankyrin 1 (TRPA1) channel, expressed by trigeminal nociceptors, is sensitive to oxidative stress and is targeted by nitric oxide or its by-products. Herein, we explored the role of TRPA1 in glyceryl trinitrate-evoked allodynia. Systemic administration of glyceryl trinitrate elicited in the mouse periorbital area an early and transient vasodilatation and a delayed and prolonged mechanical allodynia. The systemic, intrathecal or local administration of selective enzyme inhibitors revealed that nitric oxide, liberated from the parent drug by aldehyde dehydrogenase 2 (ALDH2), initiates but does not maintain allodynia. The central and the final phases of allodynia were respectively associated with generation of reactive oxygen and carbonyl species within the trigeminal ganglion. Allodynia was absent in TRPA1-deficient mice and was reversed by TRPA1 antagonists. Knockdown of neuronal TRPA1 by intrathecally administered antisense oligonucleotide and selective deletion of TRPA1 from sensory neurons in Advillin-Cre; Trpa1fl/fl mice revealed that nitric oxide-dependent oxidative and carbonylic stress generation is due to TRPA1 stimulation, and resultant NADPH oxidase 1 (NOX1) and NOX2 activation in the soma of trigeminal ganglion neurons. Early periorbital vasodilatation evoked by glyceryl trinitrate was attenuated by ALDH2 inhibition but was unaffected by TRPA1 blockade. Antagonists of the calcitonin gene-related peptide receptor did not affect the vasodilatation but partially inhibited allodynia. Thus, although both periorbital allodynia and vasodilatation evoked by glyceryl trinitrate are initiated by nitric oxide, they are temporally and mechanistically distinct. While vasodilatation is due to a direct nitric oxide action in the vascular smooth muscle, allodynia is a neuronal phenomenon mediated by TRPA1 activation and ensuing oxidative stress. The autocrine pathway, sustained by TRPA1 and NOX1/2 within neuronal cell bodies of trigeminal ganglia, may sensitize meningeal nociceptors and second order trigeminal neurons to elicit periorbital allodynia, and could be of relevance for migraine-like headaches evoked by glyceryl trinitrate in humans.

Schwann cell TRPA1 mediates neuroinflammation that sustains macrophage-dependent neuropathic pain in mice.

It is known that transient receptor potential ankyrin 1 (TRPA1) channels, expressed by nociceptors, contribute to neuropathic pain. Here we show that TRPA1 is also expressed in Schwann cells. We found that in mice with partial sciatic nerve ligation, TRPA1 silencing in nociceptors attenuated mechanical allodynia, without affecting macrophage infiltration and oxidative stress, whereas TRPA1 silencing in Schwann cells reduced both allodynia and neuroinflammation. Activation of Schwann cell TRPA1 evoked NADPH oxidase 1 (NOX1)-dependent H2O2 release, and silencing or blocking Schwann cell NOX1 attenuated nerve injury-induced macrophage infiltration, oxidative stress and allodynia. Furthermore, the NOX2-dependent oxidative burst, produced by macrophages recruited to the perineural space activated the TRPA1-NOX1 pathway in Schwann cells, but not TRPA1 in nociceptors. Schwann cell TRPA1 generates a spatially constrained gradient of oxidative stress, which maintains macrophage infiltration to the injured nerve, and sends paracrine signals to activate TRPA1 of ensheathed nociceptors to sustain mechanical allodynia.

The anti-migraine component of butterbur extracts, isopetasin, desensitizes peptidergic nociceptors by acting on TRPA1 cation channel.

BACKGROUND AND PURPOSE: The mechanism of the anti-migraine action of extracts of butterbur [Petasites hybridus (L.) Gaertn.] is unknown. Here, we investigated the ability of isopetasin, a major constituent of these extracts, to specifically target TRPA1 channel and to affect functional responses relevant to migraine. EXPERIMENTAL APPROACH: Single-cell calcium imaging and patch-clamp recordings in human and rodent TRPA1-expressing cells, neurogenic motor responses in rodent isolated urinary bladder, release of CGRP from mouse spinal cord in vitro and facial rubbing in mice and meningeal blood flow in rats were examined. KEY RESULTS: Isopetasin induced (i) calcium responses and currents in rat/mouse trigeminal ganglion (TG) neurons and in cells expressing the human TRPA1, (ii) substance P-mediated contractions of rat isolated urinary bladders and (iii) CGRP release from mouse dorsal spinal cord, responses that were selectively abolished by genetic deletion or pharmacological antagonism of TRPA1 channels. Pre-exposure to isopetasin produced marked desensitization of allyl isothiocyanate (AITC, TRPA1 channel agonist)- or capsaicin (TRPV1 channel agonist)-evoked currents in rat TG neurons, contractions of rat or mouse bladder and CGRP release from mouse central terminals of primary sensory neurons. Repeated intragastric administration of isopetasin attenuated mouse facial rubbing, evoked by local AITC or capsaicin, and dilation of rat meningeal arteries by acrolein or ethanol (TRPA1 and TRPV1 channel agonists respectively). CONCLUSION AND IMPLICATIONS: Activation of TRPA1 channels by isopetasin results in excitation of neuropeptide-containing nociceptors, followed by marked heterologous neuronal desensitization. Such atten uation in pain and neurogenic inflammation may account for the anti-migraine action of butterbur.

TRPA1 Mediates Aromatase Inhibitor-Evoked Pain by the Aromatase Substrate Androstenedione.

Aromatase inhibitors (AI) induce painful musculoskeletal symptoms (AIMSS), which are dependent upon the pain transducing receptor TRPA1. However, as the AI concentrations required to engage TRPA1 in mice are higher than those found in the plasma of patients, we hypothesized that additional factors may cooperate to induce AIMSS. Here we report that the aromatase substrate androstenedione, unique among several steroid hormones, targeted TRPA1 in peptidergic primary sensory neurons in rodent and human cells expressing the native or recombinant channel. Androstenedione dramatically lowered the concentration of letrozole required to engage TRPA1. Notably, addition of a minimal dose of androstenedione to physiologically ineffective doses of letrozole and oxidative stress byproducts produces AIMSS-like behaviors and neurogenic inflammatory responses in mice. Elevated androstenedione levels cooperated with low letrozole concentrations and inflammatory mediators were sufficient to provoke AIMSS-like behaviors. The generation of such painful conditions by small quantities of simultaneously administered TRPA1 agonists justifies previous failure to identify a precise link between AIs and AIMSS, underscoring the potential of channel antagonists to treat AIMSS. Cancer Res; 76(23); 7024-35. ©2016 AACR.