Transient Receptor Potential Ankyrin 1 Antagonist Research Articles

Macrophages and Schwann cell TRPA1 mediate chronic allodynia in a mouse model of complex regional pain syndrome type I

Complex regional pain syndrome type I (CRPS-I) is characterized by intractable chronic pain. Poor understanding of the underlying mechanisms of CRPS-I accounts for the current unsatisfactory treatment. Antioxidants and antagonists of the oxidative stress-sensitive channel, the transient receptor potential ankyrin 1 (TRPA1), have been found to attenuate acute nociception and delayed allodynia in models of CRPS-I, evoked by ischemia and reperfusion (I/R) of rodent hind limb (chronic post ischemia pain, CPIP). However, it is unknown how I/R may lead to chronic pain mediated by TRPA1. Here, we report that the prolonged (day 1–15) mechanical and cold allodynia in the hind limb of CPIP mice was attenuated permanently in Trpa1−/− mice and transiently after administration of TRPA1 antagonists (A-967079 and HC-030031) or an antioxidant (α-lipoic acid). Indomethacin treatment was, however, ineffective. We also found that I/R increased macrophage (F4/80+ cell) number and oxidative stress markers, including 4-hydroxynonenal (4-HNE), in the injured tibial nerve. Macrophage-deleted MaFIA (Macrophage Fas-Induced Apoptosis) mice did not show I/R-evoked endoneurial cell infiltration, increased 4-HNE and mechanical and cold allodynia. Furthermore, Trpa1−/− mice did not show any increase in macrophage number and 4-HNE in the injured nerve trunk. Notably, in mice with selective deletion of Schwann cell TRPA1 (Plp1-CreERT;Trpa1fl/fl mice), increases in macrophage infiltration, 4-HNE and mechanical and cold allodynia were attenuated. In the present mouse model of CRPS-I, we propose that the initial oxidative stress burst that follows reperfusion activates a feed forward mechanism that entails resident macrophages and Schwann cell TRPA1 of the injured tibial nerve to sustain chronic neuroinflammation and allodynia. Repeated treatment one hour before and for 3 days after I/R with a TRPA1 antagonist permanently protected CPIP mice against neuroinflammation and allodynia, indicating possible novel therapeutic strategies for CRPS-I.

Role of Transient Receptor Potential Ankyrin‐1 (TRPA1) in Endothelial‐Specific Effects of Diverse Aldehydes

IntroductionStructurally diverse aldehydes are found in abundance in the environment including in foods, beverages and tobacco‐derived aerosols. Some aldehydes are associated with cardiovascular disease risk especially in cigarettes smokers, however, the detailed mechanisms by which aldehydes influence cardiovascular health and disease are incompletely known.ObjectiveBecause reactive aldehydes, in general, are considered instigators of endothelium dysfunction, we explored the direct effects of a diverse set of aldehydes in isolated murine blood vessels, i.e., aorta and superior mesenteric artery (SMA).MethodsAldehydes: acetaldehyde (AA); formaldehyde (FA); cinnamaldehyde (CA); crotonaldehyde (CR); and a TRPA1 agonist, allyl isothiocyanate (AITC, mustard oil), were tested for vascular effects in isolated murine aorta and SMA using isometric myography.ResultsAll aldehydes induced endothelium‐dependent relaxation in SMA. AA (1–100 mM), FA (30–1,500 μM), CA (30–300 μM) and CR (1–100 μM) strongly and reversibly relaxed (>75% relaxation) agonist‐induced contractions. High levels of FA (1.2–1.6 mM) and CR (>300 μM) irreversibly altered vascular function in aorta and SMA. To identify the mechanism of aldehyde‐induced relaxations, isolated blood vessels were pre‐contracted with phenylephrine (PE), U46,619 (thromboxane A2 analog), or 60 mM K+ (High K) and then exposed to AA, FA, CA, CR or AITC with intact or mechanically‐impaired endothelium (air‐perfused) as well as in the absence or presence of: 1) Nω‐Nitro‐L‐arginine methyl ester hydrochloride (L‐NAME); 2) an inhibitor of guanylyl cyclase (ODQ); and, 3) a TRPA1 antagonist (A967079; 1 μM). The sensitivity (EC50) but not the efficacy (% decreased tension) of FA‐ and AA‐induced relaxations was dependent on the contractile agonist (i.e., PE < U46,619 ≤ High K) indicating multiple mechanisms. In PE pre‐contracted SMA, vasorelaxations at the lowest levels of AA, FA, CA, CR and AITC were inhibited significantly by mechanically‐disrupted endothelium, L‐NAME, ODQ, and by TRPA1 antagonist except for AA, which was TRPA1 independent. TRPA1 was co‐localized with isolectin immunofluorescent staining in SMA endothelium but not in aorta.ConclusionBecause low levels (physiological) of several aldehydes (FA, CA, CR) induce reversible and efficacious vasorelaxations via TRPA1 channels, we propose that endogenous and exogenous sources of these aldehydes may significantly impact gut (mesenteric) blood flow to augment postprandial hyperemia in aid of digestion and nutrient uptake.Support or Funding InformationResearch funding from the National Institutes of Health: GM127607, HL122676, U54HL120163.

Transient receptor potential ankyrin-1 causes rapid bronchodilation via nonepithelial PGE2.

Transient receptor potential ankyrin-1 (TRPA1) is a ligand-gated cation channel that responds to endogenous and exogenous irritants. TRPA1 is expressed on multiple cell types throughout the lungs, but previous studies have primarily focused on TRPA1 stimulation of airway sensory nerves. We sought to understand the integrated physiological airway response to TRPA1 stimulation. The TRPA1 agonists allyl isothiocyanate (AITC) and cinnamaldehyde (CINN) were tested in sedated, mechanically ventilated guinea pigs in vivo. Reproducible bronchoconstrictions were induced by electrical stimulation of the vagus nerves. Animals were then treated with intravenous AITC or CINN. AITC and CINN were also tested on isolated guinea pig and mouse tracheas and postmortem human trachealis muscle strips in an organ bath. Tissues were contracted with methacholine, histamine, or potassium chloride and then treated with AITC or CINN. Some airways were pretreated with TRPA1 antagonists, the cyclooxygenase inhibitor indomethacin, the EP2 receptor antagonist PF 04418948, or tetrodotoxin. AITC and CINN blocked vagally mediated bronchoconstriction in guinea pigs. Pretreatment with indomethacin completely abolished the airway response to TRPA1 agonists. Similarly, AITC and CINN dose-dependently relaxed precontracted guinea pig, mouse, and human airways in the organ bath. AITC- and CINN-induced airway relaxation required TRPA1, prostaglandins, and PGE2 receptor activation. TRPA1-induced airway relaxation did not require epithelium or tetrodotoxin-sensitive nerves. Finally, AITC blocked airway hyperreactivity in two animal models of allergic asthma. These data demonstrate that stimulation of TRPA1 causes bronchodilation of intact airways and suggest that the TRPA1 pathway is a potential pharmacological target for bronchodilation.

TRPA1 activation mediates nociception behaviors in a mouse model of relapsing-remitting experimental autoimmune encephalomyelitis

Central neuropathic pain is the main symptom caused by spinal cord lesion in relapsing-remitting multiple sclerosis (RRMS), but its management is still not effective. The transient receptor potential ankyrin 1 (TRPA1) is a pain detecting ion channel involved in neuropathic pain development. Thus, the aim of our study was to evaluate the role of TRPA1 in central neuropathic nociception induced by relapsing-remitting experimental autoimmune encephalomyelitis (RR-EAE) mouse model. In this model, we observed the development of similar clinical conditions of RRMS in C57BL/6 female mice through RR-EAE using MOG35–55 antigen and Quil A adjuvant. At the thirty-fifth day post-induction, C57BL/6 female mice demonstrated alteration in the RR-EAE score without motor impairment, mechanical and cold allodynia. Also, significative changes in demyelinating (Mog and olig-1) and neuroinflammatory (Iba1, Gfap and Tnfa) markers were observed, but this model did not alter Trpa1 RNA expression levels in the spinal cord. The hydrogen peroxide and 4-hydroxynonenal levels (TRPA1 agonists) were increased in RR-EAE induced mice, as well as the NADPH oxidase activity. The intragastric treatment of RR-EAE induced mice with TRPA1 antagonists (HC-030031 and A-967079) and antioxidant (α-lipoic acid and apocynin) caused an antiallodynic effect. Moreover, the intrathecal administration of TRPA1 antisense oligonucleotide, HC-030031, α-lipoic acid, and apocynin transiently attenuated mechanical and cold allodynia. Thus, TRPA1 plays a key role in the induction of neuropathic pain in this model of RR-EAE and can be a possible target for investigating the development of pain in RRMS patients.

Topical transient receptor potential ankyrin 1 antagonist treatment attenuates nociception and inflammation in an ultraviolet B radiation-induced burn model in mice

BackgroundUltraviolet B (UVB) radiation exposure promotes sunburn and thereby acute and chronic inflammatory processes, contributing to pain development and maintenance. New therapeutic alternatives are necessary because typical treatments can cause adverse effects. An attractive alternative would be to target the transient receptor potential ankyrin 1 (TRPA1), a calcium-permeable, non-selective cation channel, which is involved in a variety of inflammatory pain models. ObjectiveEvaluate the peripheral participation of TRPA1 using a topical treatment (HC030031 gel formulation; a selective TRPA1 antagonist) in nociception and inflammation caused by a UVB radiation-induced burn model in male mice (25–30 g). MethodsThe mice were anaesthetised, and just the right hind paw was exposed to UVB radiation (0.75 J/cm2). Topical treatments were applied immediately after irradiation and once a day for 8 days. ResultsHC030031 gel presented suitable pH and spreadability factor, ensuring its quality and the therapeutic effect. HC030031 0.05 % reversed UVB-induced mechanical and cold allodynia, with maximum inhibition (Imax) of 69 ± 13 % and 100 % (on day 4), respectively. HC030031 0.05 % also reduced the paw edema and MPO activity, with Imax of 77 ± 6 % (on day 5) and 69 ± 28 %, respectively. Likewise, UVB radiation increased the H2O2 levels (a TRPA1 agonist) and the Ca2+ influx in mice spinal cord synaptosomes. UVB radiation-induced Ca2+ influx was reduced by HC030031. ConclusionThese findings confirm the activation of the TRPA1 channel by UVB radiation, suggesting that topical TRPA1 antagonists can be a new strategy for the adjuvant treatment of sunburn-associated pain and inflammation.

Wood Smoke Particles Stimulate MUC5AC Overproduction by Human Bronchial Epithelial Cells Through TRPA1 and EGFR Signaling.

Mucus hypersecretion is a pathological feature of acute inflammatory and chronic obstructive pulmonary diseases. Exposure to air pollutants can be a cause of pathological mucus overproduction, but mechanisms by which different forms of air pollutants elicit this response are not fully understood. In this study, particulate matter (PM) generated from burning pine wood and other types of biomass was used to determine mechanisms by which these forms of PM stimulate mucin gene expression and secretion by primary human bronchial epithelial cells (HBECs). Biomass PM < 2.5 μm generated from pine wood and several other fuels stimulated the expression and secretion of the gel-forming glycoprotein MUC5AC by HBECs. Muc5ac gene induction was also observed in mouse airways following subacute oropharyngeal delivery of pine wood smoke PM. In HBECs, MUC5AC was also induced by the transient receptor potential ankyrin-1 (TRPA1) agonists' coniferaldehyde, a component of pine smoke PM, and allyl isothiocyanate, and was attenuated by a TRPA1 antagonist. Additionally, inhibition of epidermal growth factor receptor (EGFR/ErbB1) and the EGFR signaling partners p38 MAPK and GSK3β also prevented MUC5AC overexpression. Collectively, our results suggest that activation of TRPA1 and EGFR, paired with alterations to p38 MAPK and GSK3β activity, plays a major role in MUC5AC overproduction by bronchial epithelial cells exposed to biomass smoke PM. These results reveal specific processes for how biomass smoke PM may impact the human respiratory system and highlight potential avenues for therapeutic manipulation of lung diseases that are affected by air pollutants.

Satellite glial cells in sensory ganglia express functional transient receptor potential ankyrin 1 that is sensitized in neuropathic and inflammatory pain.

Transient receptor potential ankyrin 1 (TRPA1) is well documented as an important molecule in pain hypersensitivity following inflammation and nerve injury and in many other cellular biological processes. Here, we show that TRPA1 is expressed not only by sensory neurons of the dorsal root ganglia (DRG) but also in their adjacent satellite glial cells (SGCs), as well as nonmyelinating Schwann cells. TRPA1 immunoreactivity is also detected in various cutaneous structures of sensory neuronal terminals, including small and large caliber cutaneous sensory fibers and endings. The SGC-expressed TRPA1 is functional. Like DRG neurons, dissociated SGCs exhibit a robust response to the TRPA1-selective agonist allyl isothiocyanate (AITC) by an increase of intracellular Ca2+ concentration ([Ca2+]i). These responses are abolished by the TRPA1 antagonist HC030031 and are absent in SGCs and neurons from global TRPA1 null mice. SGCs and neurons harvested from DRG proximal to painful tissue inflammation induced by plantar injection of complete Freund’s adjuvant show greater AITC-evoked elevation of [Ca2+]i and slower recovery compared to sham controls. Similar TRPA1 sensitization occurs in both SGCs and neurons during neuropathic pain induced by spared nerve injury. Together, these results show that functional TRPA1 is expressed by sensory ganglia SGCs, and TRPA1 function in SGCs is enhanced after both peripheral inflammation and nerve injury, and suggest that TRPA1 in SGCs may contribute to inflammatory and neuropathic pain.

Analysis of TRPA1 antagonist, A-967079, in plasma using high-performance liquid chromatography tandem mass-spectrometry

The noxious effects from exposure to toxic inhalation hazards (TIHs, such as isocyanates, chlorine, etc.) are known to be triggered by the activation of transient receptor potential ankyrin 1 (TRPA1) ion channel. Antagonists of TRPA1 have shown near complete attenuation of the noxious effects from TIH exposure. One of the TRPA1 antagonists, (1E,3E)-1-(4-fluorophenyl)-2-methyl-1-pentene-3-one oxime (A-967079), has shown impressive efficacy, high selectivity, high potency, and oral bioavailability. Although a validated method to quantify A-967079 in biological matrices is vital for the further development of A-967079 as a therapeutic agent, no method for its analysis from any matrix is currently available. Hence, a rapid and simple HPLC-MS/MS method was developed and validated to quantify A-967079 in rabbit plasma. The method presented here features an excellent LOD of 25 nM and a wide linear range (0.05–200 μM), with good accuracy and precision (100 ± 10.5% and <14.2% relative standard deviation, respectively). The stability of A-967079 in plasma was excellent for most of the storage conditions evaluated. The method was successfully applied to determine A-967079 from treated animals and it may facilitate the development of this TRPA1 antagonist as a therapeutic agent against the noxious effects of TIH exposure.

Role of transient receptor potential ankyrin 1 (TRPA1) on nociception caused by a murine model of breast carcinoma

Breast carcinoma causes severe pain, which decreases the quality of life of patients. Current treatments produce adverse effects and have limited efficacy. Transient potential receptor ankyrin 1 (TRPA1) is related to the onset of cancer and neuropathic pain. The aim of this study was to evaluate the involvement of TRPA1 in a model of breast carcinoma. We injected 4T1 cells in the fourth caudal mammary fat pad of female BALB/c mice, and after 20 days we observed mechanical and cold allodynia and spontaneous nociception behavior (mouse grimace scale detection, MGS). TRPA1 selective antagonist (HC-030031 or A-967079) administration or intrathecal administration of TRPA1 antisense (AS) oligonucleotide was performed. The activity of NADPH oxidase, superoxide dismutase (SOD) and hydrogen peroxide (H2O2) levels were evaluated. The chemical hyperalgesia produced by a TRPA1 agonist (allyl isothiocyanate, AITC) was also detected. The administration of TRPA1 antagonists, TRPA1 AS, or antioxidant, transiently attenuated MGS, or mechanical and cold allodynia. Intraplantar injection of AITC also caused nociception. NADPH oxidase or SOD activity and H2O2 levels were increased in the sciatic nerve and hind paw skin samples. The 4T1 cells did not express TRPA1, and the use of HC-030031 or α-lipoic acid did not reduce the cytotoxic effect of a chemotherapeutic drug (paclitaxel). Thus, TRPA1 could be investigated as a target for breast carcinoma pain treatment.

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.

TRPA1 and issues relating to animal model selection for extrapolating toxicity data to humans.

The transient receptor potential ankyrin 1 (TRPA1) ion channel is a sensor for irritant chemicals, has ancient lineage, and is distributed across animal species including humans, where it features in many organs. Its activation by a diverse panel of electrophilic molecules (TRPA1 agonists) through electrostatic binding and/or covalent attachment to the protein causes the sensation of pain. This article reviews the species differences between TRPA1 channels and their responses, to assess the suitability of different animals to model the effects of TRPA1-activating electrophiles in humans, referring to common TRPA1 activators (exogenous and endogenous) and possible mechanisms of action relating to their toxicology. It concludes that close matching of in vitro and in vivo models will help optimise the identification of relevant biochemical and physiological responses to benchmark the efficacy of potential therapeutic drugs, including TRPA1 antagonists, to counter the toxic effects of those electrophiles capable of harming humans. The analysis of the species issue provided should aid the development of medical treatments to counter poisoning by such chemicals.

Computational Drug Repurposing Algorithm Targeting TRPA1 Calcium Channel as a Potential Therapeutic Solution for Multiple Sclerosis.

Multiple sclerosis (MS) is a chronic autoimmune disease affecting the central nervous system (CNS) through neurodegeneration and demyelination, leading to physical/cognitive disability and neurological defects. A viable target for treating MS appears to be the Transient Receptor Potential Ankyrin 1 (TRPA1) calcium channel, whose inhibition has been shown to have beneficial effects on neuroglial cells and protect against demyelination. Using computational drug discovery and data mining methods, we performed an in silico screening study combining chemical graph mining, quantitative structure–activity relationship (QSAR) modeling, and molecular docking techniques in a global prediction model in order to identify repurposable drugs as potent TRPA1 antagonists that may serve as potential treatments for MS patients. After screening the DrugBank database with the combined generated algorithm, 903 repurposable structures were selected, with 97 displaying satisfactory inhibition probabilities and pharmacokinetics. Among the top 10 most probable inhibitors of TRPA1 with good blood brain barrier (BBB) permeability, desvenlafaxine, paliperidone, and febuxostat emerged as the most promising repurposable agents for treating MS. Molecular docking studies indicated that desvenlafaxine, paliperidone, and febuxostat are likely to induce allosteric TRPA1 channel inhibition. Future in vitro and in vivo studies are needed to confirm the biological activity of the selected hit molecules.

Bladder Dysfunction in an Obese Zucker Rat: The Role of TRPA1 Channels, Oxidative Stress, and Hydrogen Sulfide.

Purpose This study investigates whether functionality and/or expression changes of transient receptor potential vanilloid 1 (TRPV1) and transient receptor potential ankyrin 1 (TRPA1) channels, oxidative stress, and hydrogen sulfide (H2S) are involved in the bladder dysfunction from an insulin-resistant obese Zucker rat (OZR). Materials and Methods Detrusor smooth muscle (DSM) samples from the OZR and their respective controls, a lean Zucker rat (LZR), were processed for immunohistochemistry for studying the expression of TRPA1 and TRPV1 and the H2S synthase cystathionine beta-synthase (CBS) and cysthathionine-γ-lyase (CSE). Isometric force recordings to assess the effects of TRPA1 agonists and antagonists on DSM contractility and measurement of oxidative stress and H2S production were also performed. Results Neuronal TRPA1 expression was increased in the OZR bladder. Electrical field stimulation- (EFS-) elicited contraction was reduced in the OZR bladder. In both LZR and OZR, TRPA1 activation failed to modify DSM basal tension but enhanced EFS contraction; this response is inhibited by the TRPA1 blockade. In the OZR bladder, reactive oxygen species, malondialdehyde, and protein carbonyl contents were increased and antioxidant enzyme activities (superoxide dismutase, catalase, GR, and GPx) were diminished. CSE expression and CSE-generated H2S production were also reduced in the OZR. Both TRPV1 and CBS expressions were not changed in the OZR. Conclusions These results suggest that an increased expression and functionality of TRPA1, an augmented oxidative stress, and a downregulation of the CSE/H2S pathway are involved in the impairment of nerve-evoked DSM contraction from the OZR.

Knockout of TRPA1 exacerbates angiotensin II-induced kidney injury

Macrophage-mediated inflammation plays a critical role in hypertensive kidney disease. Here, we investigated the role of transient receptor potential ankyrin 1 (TRPA1), a sensor of inflammation, in angiotensin II (ANG II)-induced renal injury. Subcutaneous infusion of ANG II (600 ng·min-1·kg-1) for 28 days was used to induce hypertension and renal injury in mice. The results showed that ANG II-induced hypertensive mice have decreased renal Trpa1 expression (P < 0.01), whereas ANG II receptor type 1a-deficient hypotensive mice have increased renal Trpa1 expression (P < 0.05) compared with their normotensive counterparts. ANG II induced similar elevations of systolic blood pressure in Trpa1-/- and wild-type (WT) mice but led to higher levels of blood urea nitrogen (P < 0.05), serum creatinine (P < 0.05), and renal fibrosis (P < 0.01) in Trpa1-/- mice than WT mice. Similarly, ANG II increased both CD68+/inducible nitric oxide synthase+ M1 and CD68+/arginase 1+ M2 macrophages in the kidneys of both Trpa1-/- and WT mice (all P < 0.01), with higher extents in Trpa1-/- mice (both P < 0.01). Compared with WT mice, Trpa1-/- mice had significantly increased expression levels of inflammatory cytokines and their receptors in the kidney. Cultured murine macrophages were stimulated with phorbol 12-myristate 13-acetate, which downregulated gene expression of TRPA1 (P < 0.01). A TRPA1 agonist, cinnamaldehyde, significantly inhibited phorbol 12-myristate 13-acetate-stimulated expression of IL-1β and chemokine (C-C motif) ligand 2 in macrophages, which were attenuated by pretreatment with a TRPA1 antagonist, HC030031. Furthermore, activation of TRPA1 with cinnamaldehyde induced apoptosis of macrophages. These findings suggest that TRPA1 may play a protective role in ANG II-induced renal injury, likely through inhibiting macrophage-mediated inflammation.

Effects of Cinnamaldehyde on Smooth Muscle Preparations

The effects of cinnamaldehyde (CNA), known as a transient receptor potential ankyrin 1 (TRPA1) agonist, on guinea-pig ileum and urinary bladder were studied in isolated organ experiments. Contractile effects were found to be present on both preparations. In the ileum, both cholinergic and purinergic (pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid tetrasodium salt-sensitive) mechanisms are involved; the TRPA1 antagonist A967079 (1 µmol/L) significantly reduced the response. The contractile response to CNA in the bladder, but not in the ileum, was significantly reduced by in vitro capsaicin desensitization. In the bladder A967079 or the TRPV1 antagonist, BCTC failed to reduce the response. A direct relaxation on the smooth muscle was detected in the precontracted ileum. In the precontracted urinary bladder, CNA also caused relaxation that was insensitive to capsaicin pretreatment. It is suggested that CNA excites the muscles of the bladder via activation of capsaicin-sensitive nerves; in the ileum, it may interact with TRPA1 located on tissue elements that initiate both purinergic and cholinergic mechanisms. The relaxant effects of CNA may be due to the direct inhibition of the smooth muscles.

Carboplatin Enhances the Activity of Human Transient Receptor Potential Ankyrin 1 through the Cyclic AMP-Protein Kinase A-A-Kinase Anchoring Protein (AKAP) Pathways.

Carboplatin, an anticancer drug, often causes chemotherapy-induced peripheral neuropathy (PN). Transient receptor potential ankyrin 1 (TRPA1), a non-selective cation channel, is a polymodal nociceptor expressed in sensory neurons. TRPA1 is not only involved in pain transmission, but also in allodynia or hyperalgesia development. However, the effects of TRPA1 on carboplatin-induced PN is unclear. We revealed that carboplatin induced mechanical allodynia and cold hyperalgesia, and the pains observed in carboplatin-induced PN models were significantly suppressed by the TRPA1 antagonist HC-030031 without a change in the level of TRPA1 protein. In cells expressing human TRPA, carboplatin had no effects on changes in intracellular Ca2+ concentration ([Ca2+]i); however, carboplatin pretreatment enhanced the increase in [Ca2+]i induced by the TRPA1 agonist, allyl isothiocyanate (AITC). These effects were suppressed by an inhibitor of protein kinase A (PKA). The PKA activator forskolin enhanced AITC-induced increase in [Ca2+]i and carboplatin itself increased intracellular cyclic adenosine monophosphate (cAMP) levels. Moreover, inhibition of A-kinase anchoring protein (AKAP) significantly decreased the carboplatin-induced enhancement of [Ca2+]i induced by AITC and improved carboplatin-induced mechanical allodynia and cold hyperalgesia. These results suggested that carboplatin induced mechanical allodynia and cold hyperalgesia by increasing sensitivity to TRPA1 via the cAMP-PKA-AKAP pathway.

TRPA1 Channel is Involved in SLIGRL-Evoked Thermal and Mechanical Hyperalgesia in Mice.

Persistent itch (pruritus) accompanying dermatologic and systemic diseases can significantly impair the quality of life. It is well known that itch is broadly categorized as histaminergic (sensitive to antihistamine medications) or non-histaminergic. Sensory neurons expressing Mas-related G-protein-coupled receptors (Mrgprs) mediate histamine-independent itch. These receptors have been shown to bind selective pruritogens in the periphery and mediate non-histaminergic itch. For example, mouse MrgprA3 responds to chloroquine (an anti-malarial drug), and are responsible for relaying chloroquine-induced scratching in mice. Mouse MrgprC11 responds to a different subset of pruritogens including bovine adrenal medulla peptide (BAM8–22) and the peptide Ser-Leu-Ile-Gly-Arg-Leu (SLIGRL). On the other hand, the possibility that itch mediators also influence pain is supported by recent findings that most non-histaminergic itch mediators require the transient receptor potential ankyrin 1 (TRPA1) channel. We have recently found a significant increase of thermal and mechanical hyperalgesia induced by non-histaminergic pruritogens chloroquine and BAM8–22, injected into mice hindpaw, for the first 30–45 min. Pretreatment with TRPA1 channel antagonist HC-030031 did significantly reduce the magnitude of this hyperalgesia, as well as significantly shortened the time-course of hyperalgesia induced by chloroquine and BAM8–22. Here, we report that MrgprC11-mediated itch by their agonist SLIGRL is accompanied by heat and mechanical hyperalgesia via the TRPA1 channel. We measured nociceptive thermal paw withdrawal latencies and mechanical thresholds bilaterally in mice at various time points following intra-plantar injection of SLIGRL producing hyperalgesia. When pretreated with the TRPA1 antagonist HC-030031, we found a significant reduction of thermal and mechanical hyperalgesia.

Transient receptor potential ankyrin 1 (TRPA1) as a factor and drug target in osteoarthritis: TRPA1 mediates fibroblast growth factor 2 expression in chondrocytes

Purpose: Transient receptor potential ankyrin 1 (TRPA1) is a membrane-associated cation channel, which is known to be involved in nociception and neurogenic inflammation. TRPA1 is widely expressed in neurons, and according to more recent findings, also in non-neuronal cells, including synoviocytes and keratinocytes. TRPA1 acts as a chemosensor of pungent environmental compounds, but it is also activated by agents formed endogenously in inflammatory and hypoxic conditions such as those found in osteoarthritic joints. We have recently shown in monosodium iodoacetate-induced experimental osteoarthritis that TRPA1 activation mediates inflammation, cartilage degradation and pain (Moilanen et al. Osteoarthritis Cartilage 2015). We have also shown that TRPA1 is functionally expressed in human chondrocytes, where it mediates inflammatory effects (Nummenmaa et al. Arthritis Res Ther 2016). Further, we found that anti-inflammatory drugs dexamethasone and aurothiomalate downregulate the expression of TRPA1 in human chondrocytes (Nummenmaa et al. RMD Open 2017), revealing TRPA1 as a potential mediator and drug target in osteoarthritis. Fibroblast growth factor 2 (FGF-2) belongs to the fibroblast growth factor family, which is a large group of molecules involved in the regulation of connective tissue development and metabolism. FGF-2 is found in the synovial fluid and cartilage of osteoarthritis patients, and has previously been associated with the pathogenesis of osteoarthritis. In the present study, we investigated the functional consequences of TRPA1 activation in chondrocytes focusing in particularly on the expression of FGF-2. Methods: Chondrocytes were isolated from cartilage tissue from wild type (WT) and TRPA1 deficient (knock-out, KO) mice and cultured in the absence or presence of IL-1β. Total RNA was isolated and genome-wide expression analysis was carried out with next generation RNA sequencing (NGS) method with Illumina HiSeq2500 at the Institute for Molecular Medicine Finland. Results from the RNA sequencing analysis were verified by quantitative RT-PCR. Primary human OA chondrocytes were isolated from cartilage samples obtained from patients undergoing knee replacement surgery. Chondrocytes were cultured in the presence of the TRPA1-inducing cytokine IL-1β alone and together with the selective TRPA1 antagonist HC-030031. For experiments investigating the effects of FGF-2 in chondrocytes, the cells were cultured in the presence of FGF-2. Expression of FGF-2 subsequent to TRPA1 activation was measured by quantitative RT-PCR, and aggrecan, collagen II and MMP-enzymes by quantitative RT-PCR/immunoassay. Results: The expression of FGF-2 was significantly downregulated in chondrocytes from TRPA KO mice compared to WT mice. The downregulation of FGF-2 was verified by performing quantitative RT-PCR analysis on the sequenced samples, and on a larger set of corresponding samples. The results were confirmed using primary human OA chondrocytes. Pharmacological inhibition of TRPA1 with the selective antagonist HC-030031 downregulated FGF-2 expression in these cells. We further investigated the effects of FGF-2 in human OA chondrocytes, and found that FGF-2 upregulated the production of cartilage matrix degrading enzymes MMP-1 and MMP-13, and downregulated the expression of cartilage matrix components aggrecan and collagen II. Conclusions: The results revealed a hitherto unknown role for TRPA1 in the regulation of FGF-2 expression in chondrocytes. Further, FGF-2 was confirmed to have catabolic and anti-anabolic effects in human OA chondrocytes. Inhibition of TRPA1 and subsequent downregulation of FGF-2 could therefore have beneficial effects on the balance of catabolic and anabolic mediators within OA cartilage. These results together with our recent data discussed above support the concept of TRPA1 as a potential mediator and drug target in osteoarthritis.

TRPA1 channel contributes to myocardial ischemia-reperfusion injury.

Myocardial ischemia-reperfusion (I/R) results in the generation of free radicals, accumulation of lipid peroxidation-derived unsaturated aldehydes, variable angina (pain), and infarction. The transient receptor potential ankyrin 1 (TRPA1) mediates pain signaling and is activated by unsaturated aldehydes, including acrolein and 4-hydroxynonenal. The contribution of TRPA1 (a Ca2+-permeable channel) to I/R-induced myocardial injury is unknown. We tested the hypothesis that cardiac TRPA1 confers myocyte sensitivity to aldehyde accumulation and promotes I/R injury. Although basal cardiovascular function in TRPA1-null mice was similar to that in wild-type (WT) mice, infarct size was significantly smaller in TRPA1-null mice than in WT mice (34.1 ± 9.3 vs. 14.3 ± 9.9% of the risk region, n = 8 and 7, respectively, P < 0.05), despite a similar I/R-induced area at risk (40.3 ±8.4% and 42.2 ± 11.3% for WT and TRPA1-null mice, respectively) after myocardial I/R (30 min of ischemia followed by 24 h of reperfusion) in situ. Positive TRPA1 immunofluorescence was present in murine and human hearts and was colocalized with connexin43 at intercalated disks in isolated murine cardiomyocytes. Cardiomyocyte TRPA1 was confirmed by quantitative RT-PCR, DNA sequencing, Western blot analysis, and electrophysiology. A role of TRPA1 in cardiomyocyte toxicity was demonstrated in isolated cardiomyocytes exposed to acrolein, an I/R-associated toxin that induces Ca2+ accumulation and hypercontraction, effects significantly blunted by HC-030031, a TRPA1 antagonist. Protection induced by HC-030031 was quantitatively equivalent to that induced by SN-6, a Na+/Ca2+ exchange inhibitor, further supporting a role of Ca2+ overload in acrolein-induced cardiomyocyte toxicity. These data indicate that cardiac TRPA1 activation likely contributes to I/R injury and, thus, that TRPA1 may be a novel therapeutic target for decreasing myocardial I/R injury. NEW & NOTEWORTHY Transient receptor potential ankyrin 1 (TRPA1) activation mediates increased blood flow, edema, and pain reception, yet its role in myocardial ischemia-reperfusion (I/R) injury is unknown. Genetic ablation of TRPA1 significantly decreased myocardial infarction after I/R in mice. Functional TRPA1 in cardiomyocytes was enriched in intercalated disks and contributed to acrolein-induced Ca2+ overload and hypercontraction. These data indicate that I/R activation of TRPA1 worsens myocardial infarction; TRPA1 may be a potential target to mitigate I/R injury.

Transient Receptor Potential Ankyrin 1 Enhances Ovalbumin-Induced Acute Allergic Inflammation in Murine Models

Background: Transient receptor potential ankyrin 1 (TRPA1) is an ion channel known to mediate nociception and neurogenic inflammation, and to be activated by reactive oxygen and nitrogen species (ROS and RNS) produced at the sites of inflammation. Because neurogenic inflammation as well as the release of ROS and RNS are typical features of early stages of allergic responses, we hypothesized that TRPA1 may be involved in triggering and/or amplifying allergic inflammation. Objective: This study aims at exploring the role of TRPA1 ion channel in acute ovalbumin-induced allergic inflammation in applicable murine models. Methods: The effects of pharmacological blockade and genetic deletion of TRPA1 in ovalbumin-induced allergic conjunctivitis and acute paw inflammation were studied in mice sensitized to ovalbumin. Results: Ovalbumin-induced allergic conjunctivitis was milder in TRPA1-deficient mice and alleviated in wild-type mice treated with the TRPA1 antagonist TCS 5861528. Subcutaneous challenge with ovalbumin caused a significant paw edema and interleukin (IL)-4 production in sensitized mice; these responses were attenuated in animals treated with the TRPA1 antagonist and in TRPA1-deficient mice. Interestingly, blockade of the major secondary effector of TRPA1, substance P, also resulted in attenuated ovalbumin-induced paw edema and IL-4 production. However, the splenocytes’ responses to ovalbumin were similar in cells from wild-type and TRPA1-deficient mice sensitized to ovalbumin. Conclusion: These results introduce a novel concept that TRPA1 mediates early events in allergic inflammation, but does not seem to affect allergic sensitization, and could therefore be a novel drug target to treat conditions associated with allergic inflammation.