Inhibition Of Transient Receptor Potential Ankyrin 1 Research Articles

The peptide Phα1β, from spider venom, acts as a TRPA1 channel antagonist with antinociceptive effects in mice.

Peptides from venomous animals have long been important for understanding pain mechanisms and for the discovery of pain treatments. Here, we hypothesized that Phα1β, a peptide from the venom of the armed spider Phoneutria nigriventer, produces analgesia by blocking the TRPA1 channel. Cultured rat dorsal root ganglion (DRG) neurons, human fetal lung fibroblasts (IMR90) or HEK293 cells expressing the human TRPA1 (hTRPA1-HEK293), human TRPV1 (hTRPV1-HEK293) or human TRPV4 channels (hTRPV4-HEK293), were used for calcium imaging and electrophysiology. Nociceptive responses induced by TRPA1, TRPV1 or TRPV4 agonists or by bortezomib were investigated in mice. Phα1β selectively inhibited calcium responses and currents evoked by the TRPA1 agonist, allyl isothiocyanate (AITC), on hTRPA1-HEK293, IMR90 fibroblasts and DRG neurons. Phα1β did not affect calcium responses evoked by selective TRPV1 (capsaicin) or TRPV4 (GSK 1016790A) agonists on the various cell types. Intrathecal (i.t.) and intraplantar (i.pl.) administration of low doses of Phα1β (up to 300pmol per paw) attenuated acute nociception and mechanical and cold hyperalgesia evoked by AITC (i.t. or i.pl.), without affecting responses produced by capsaicin or hypotonic solution. Notably, Phα1β abated the TRPA1-dependent neuropathic pain-like responses induced by bortezomib. In vitro and in vivo inhibition of TRPA1 by Phα1β was reproduced by a recombinant form of the peptide, CTK 01512-2. Phα1β and CTK 01512-2 selectively target TRPA1, but not other TRP channels. This specific action underlines the potential of Phα1β and CTK 01512-2 for pain treatment.

Transient receptor potential ankyrin 1 (TRPA1) is functionally expressed in primary human osteoarthritic chondrocytes.

BackgroundTransient receptor potential ankyrin 1 (TRPA1) is a membrane-associated cation channel, widely expressed in neuronal cells and involved in nociception and neurogenic inflammation. We showed recently that TRPA1 mediates cartilage degradation and joint pain in the MIA-model of osteoarthritis (OA) suggesting a hitherto unknown role for TRPA1 in OA. Therefore, we aimed to investigate whether TRPA1 is expressed and functional in human OA chondrocytes.MethodsExpression of TRPA1 in primary human OA chondrocytes was assessed by qRT-PCR and Western blot. The functionality of the TRPA1 channel was assessed by Ca2+-influx measurements. Production of MMP-1, MMP-3, MMP-13, IL-6, and PGE2 subsequent to TRPA1 activation was measured by immunoassay.ResultsWe show here for the first time that TRPA1 is expressed in primary human OA chondrocytes and its expression is increased following stimulation with inflammatory factors IL-1β, IL-17, LPS, and resistin. Further, the TRPA1 channel was found to be functional, as stimulation with the TRPA1 agonist AITC caused an increase in Ca2+ influx, which was attenuated by the TRPA1 antagonist HC-030031. Genetic depletion and pharmacological inhibition of TRPA1 downregulated the production of MMP-1, MMP-3, MMP-13, IL-6, and PGE2 in osteoarthritic chondrocytes and murine cartilage, respectively.ConclusionsThe TRPA1 cation channel was found to be functionally expressed in primary human OA chondrocytes, which is an original finding. The presence and inflammatory and catabolic effects of TRPA1 in human OA chondrocytes propose a highly intriguing role for TRPA1 as a pathogenic factor and drug target in OA.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-016-1080-4) contains supplementary material, which is available to authorized users.

Monosodium iodoacetate-induced inflammation and joint pain are reduced in TRPA1 deficient mice – potential role of TRPA1 in osteoarthritis

Intra-articularly injected monosodium iodoacetate (MIA) induces joint pathology mimicking osteoarthritis (OA) and it is a widely used experimental model of OA. MIA induces acute inflammation, cartilage degradation and joint pain. Transient Receptor Potential Ankyrin 1 (TRPA1) is an ion channel known to mediate nociception and neurogenic inflammation. Here, we tested the hypothesis that TRPA1 would be involved in the development of MIA-induced acute inflammation, cartilage changes and joint pain. The effects of pharmacological blockade (by TCS 5861528) and genetic depletion of TRPA1 were studied in MIA-induced acute paw inflammation. Cartilage changes (histological scoring) and joint pain (weight-bearing test) in MIA-induced experimental OA were compared between wild type and TRPA1 deficient mice. The effects of MIA were also studied in primary human OA chondrocytes and in mouse cartilage. MIA evoked acute inflammation, degenerative cartilage changes and joint pain in wild type mice. Interestingly, these responses were attenuated in TRPA1 deficient animals. MIA-induced paw inflammation was associated with increased tissue levels of substance P; and the inflammatory edema was reduced by pretreatment with catalase, with the TRPA1 antagonist TCS 5861528 and with the neurokinin 1 receptor antagonist L703,606. In chondrocytes, MIA enhanced interleukin-1 induced cyclooxygenase-2 (COX-2) expression, an effect that was blunted by pharmacological inhibition and genetic depletion of TRPA1. TRPA1 was found to mediate acute inflammation and the development of degenerative cartilage changes and joint pain in MIA-induced experimental OA in the mouse. The results reveal TRPA1 as a potential mediator and drug target in OA.

A role for CC-chemokine receptor 7 (CCR7) in a murine model of osteoarthritis: Impact on joint structure and function

A role for CC-chemokine receptor 7 (CCR7) in a murine model of osteoarthritis: Impact on joint structure and function

S3-4 H2S targets transient receptor potential receptors to relieve from inflammation and pain

Transient Receptor Potential Vanilloid 4 (TRPV4) and Transient Receptor Potential Ankyrin 1 (TRPA1) are present both in sensory neurons and in intestinal epithelial cells, where their activation causes intracellular calcium mobilization. Activation of each receptor in sensory neurons conveys pro-nociceptive signals, while in intestinal epithelial cells, their activation confers a pro-inflammatory phenotype with chemokine release. Further, TRPV4 or TRPA1 activation in mouse intestine induced visceral hyperalgesia and colitis, confirming that these channels could participate to intestinal pain and inflammation. We hypothesized that H 2 S is able to regulate TRPV4 and TRPA1functions in intestinal epithelial cells and sensory neurons. Methods TRPV4 and TRPA1 responses were evaluated by measuring calcium flux with a Fluo-8 probe in HEK cells transfected with TRPV4 or with TRPA1, but also in normal derived Colon Mucosa (NCM460) cells, Caco-2 cells (an intestinal epithelium cell line) and mouse DRG (dorsal ganglion roots) primary neurons. Cells were pre-exposed to Sodium Hydrogen Sulfide (NaHS) a H 2 S donor, or their vehicle, 3-min before a second stimulation with the specific agonist of TRPV4, 4 α PDD (4 α -phorbol-12,13-didenoate), the agonist of TRPA1 allyl isothiocyanate (AITC) or vehicle. IL-8 expression was also evaluated after 6 h of treatment with NaHS and TRPV4 or TRPA1 stimulation. Results 4 α PDD or AITC but not NaHS increased the intracellular calcium concentration respectively in HEK TRPV4 and HEK TRPA1. TRPV4 or TRPA1 agonist induced a dose-dependent calcium flux in NCM460 cells, Caco-2 cells and neurons. Incubation of NCM460 and Caco-2 cells with NaHS (200–1000 μM) dose-dependently decreased the calcium response to a pro-inflammatory dose of TRPV4 or TRPA1 agonist (50 μM). In neurons, the calcium response to 25 μM of 4 α PDD or AITC was also significantly reduced with a NaHS pre-treatment (1 mM). Furthermore, IL-8 expression was decreased when NCM460 and Caco-2 cells were pre-treated with NaHS. Conclusions NaHS exposure decreased cellular response to TRPV4 or TRPA1 agonist. Considering the pro-inflammatory and pro-nociceptive effects of TRPV4 or TRPA1 activation in the gut, H 2 S could exert there, anti-inflammatory effects through the inhibition of TRPV4 and TRPA1. Hydrogen sulfide efficiently reduces visceral inflammation and pain as previously demonstrated, our results suggest that these protective effects of H2S donnors could be mediated by TRPV4 and TRPA1 inhibition.

TRPA1-Dependent Pruritus in IL-13–Induced Chronic Atopic Dermatitis

Chronic debilitating pruritus is a cardinal feature of atopic dermatitis (AD). Little is known about the underlying mechanisms. Antihistamines lack efficacy in treating itch in AD, suggesting the existence of histamine-independent itch pathways in AD. Transient receptor potential ankyrin 1 (TRPA1) is essential in the signaling pathways that promote histamine-independent itch. In this study, we tested the hypothesis that TRPA1-dependent neural pathways play a key role in chronic itch in AD using an IL-13-transgenic mouse model of AD. In these mice, IL-13 causes chronic AD characterized by intensive chronic itch associated with markedly enhanced growth of dermal neuropeptide-secreting afferent nerve fibers and enhanced expression of TRPA1 in dermal sensory nerve fibers, their dorsal root ganglia, and mast cells. Inhibition of TRPA1 with a specific antagonist in these mice selectively attenuated itch-evoked scratching. Genetic deletion of mast cells in these mice led to significantly diminished itch-scratching behaviors and reduced TRPA1 expression in dermal neuropeptide containing afferents in the AD skin. Interestingly, IL-13 strongly stimulates TRPA1 expression, which is functional in calcium mobilization in mast cells. In accordance with these observations in the AD mice, TRPA1 expression was highly enhanced in the dermal afferent nerves, mast cells, and the epidermis in the lesional skin biopsies from patients with AD, but not in the skin from healthy subjects. These studies demonstrate a novel neural mechanism underlying chronic itch in AD and highlight the complex interactions among TRPA1(+) dermal afferent nerves and TRPA1(+) mast cells in a Th2-dominated inflammatory environment.

Modulation of Transient Receptor Vanilloid 1 Activity by Transient Receptor Potential Ankyrin 1

Transient receptor potential vanilloid 1 (TRPV1) is a nonselective ligand-gated cation channel responding to noxious heat, protons, and chemicals such as capsaicin. TRPV1 is expressed in sensory neurons and plays a critical role in pain associated with tissue injury, inflammation, and nerve lesions. Transient receptor potential ankyrin 1 (TRPA1) is coexpressed with TRPV1. It is activated by compounds that cause a burning sensation (e.g., mustard oil) and, indirectly, by components of the inflammatory milieu eliciting nociceptor excitation and pain hypersensitivity. Previous studies indicate an interaction of TRPV1 and TRPA1 signaling pathways. Here we sought to examine the molecular mechanisms underlying such interactions in nociceptive neurons. We first excluded physical interactions of both channels using radioligand binding studies. By microfluorimetry, electrophysiological experiments, cAMP measurements, and site-directed mutagenesis we found a sensitization of TRPV1 after TRPA1 stimulation with mustard oil in a calcium and cAMP/protein kinase A (PKA)-dependent manner. TRPA1 stimulation enhanced TRPV1 phosphorylation via the putative PKA phosphorylation site serine 116. We also detected calcium-sensitive increased TRPV1 activity after TRPA1 activation in dorsal root ganglion neurons. The inhibition of TRPA1 by HC-030031 (1,2,3,6-tetrahydro-1,3-dimethyl-N-[4-(1-methylethyl)phenyl]-2,6-dioxo-7H-purine-7-acetamide, 2-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-7H-purin-7-yl)-N-(4-isopropylphenyl)acetamide) after its initial stimulation (and the calcium-insensitive TRPA1 mutant D477A) still showed increased capsaicin-induced TRPV1 activity. This excludes a calcium-induced additive TRPA1 current after TRPV1 stimulation. Our study shows sensitization of TRPV1 via activation of TRPA1, which involves adenylyl cyclase, increased cAMP, subsequent translocation and activation of PKA, and phosphorylation of TRPV1 at PKA phosphorylation residues. This suggests that cross-sensitization of TRP channels contributes to enhanced pain sensitivity in inflamed tissues.

Transient receptor potential ankyrin-1 (TRPA1) modulates store-operated Ca2 + entry by regulation of STIM1-Orai1 association

TRPA1 is a non-selective Ca2+ permeable channel located in the plasma membrane that functions as a cellular sensor detecting mechanical, chemical and thermal stimuli, being a component of neuronal, epithelial, blood and smooth muscle tissues. TRPA1 has been shown to influence a broad range of physiological processes that involve Ca2+-dependent signaling pathways. Here we report that TRPA1 is expressed in MEG01 but not in platelets at the protein level. MEG01 cells maturation induced by PMA results in attenuation of TRPA1 protein expression and enhances thapsigargin-evoked Ca2+ entry without altering the release of Ca2+ from intracellular stores. Inhibition of TRPA1 by HC-030031 results in enhancement of both thrombin- and thapsigargin-stimulated Ca2+ entry. Co-immunoprecipitation experiments revealed that TRPA1 associates with STIM1, as well as Orai1, TRPC1 and TRPC6. Downregulation of TRPA1 expression by MEG01 maturation, as well as pharmacological inhibition of TRPA1 by HC-030031, results in enhancement of the association between STIM1 and Orai1. Altogether, these findings provide evidence for a new and interesting function of TRPA1 in cellular function associated to the regulation of agonist-induced Ca2+ entry by the modulation of STIM1/Orai1 interaction.

Analysis of Transient Receptor Potential Ankyrin 1 (TRPA1) in Frogs and Lizards Illuminates Both Nociceptive Heat and Chemical Sensitivities and Coexpression with TRP Vanilloid 1 (TRPV1) in Ancestral Vertebrates

Transient receptor potential ankyrin 1 (TRPA1) and TRP vanilloid 1 (V1) perceive noxious temperatures and chemical stimuli and are involved in pain sensation in mammals. Thus, these two channels provide a model for understanding how different genes with similar biological roles may influence the function of one another during the course of evolution. However, the temperature sensitivity of TRPA1 in ancestral vertebrates and its evolutionary path are unknown as its temperature sensitivities vary among different vertebrate species. To elucidate the functional evolution of TRPA1, TRPA1s of the western clawed (WC) frogs and green anole lizards were characterized. WC frog TRPA1 was activated by heat and noxious chemicals that activate mammalian TRPA1. These stimuli also activated native sensory neurons and elicited nocifensive behaviors in WC frogs. Similar to mammals, TRPA1 was functionally co-expressed with TRPV1, another heat- and chemical-sensitive nociceptive receptor, in native sensory neurons of the WC frog. Green anole TRPA1 was also activated by heat and noxious chemical stimulation. These results suggest that TRPA1 was likely a noxious heat and chemical receptor and co-expressed with TRPV1 in the nociceptive sensory neurons of ancestral vertebrates. Conservation of TRPV1 heat sensitivity throughout vertebrate evolution could have changed functional constraints on TRPA1 and influenced the functional evolution of TRPA1 regarding temperature sensitivity, whereas conserving its noxious chemical sensitivity. In addition, our results also demonstrated that two mammalian TRPA1 inhibitors elicited different effect on the TRPA1s of WC frogs and green anoles, which can be utilized to clarify the structural bases for inhibition of TRPA1.

Apport de l’imagerie fonctionnelle à la compréhension des mécanismes de la douleur

Exaggerated responses to inhaled irritant chemicals are a hallmark of human airway diseases such as asthma. While the inflammatory processes and structural remodeling caused by respiratory irritants have received considerable attention, the mechanisms responsible for triggering acute episodes of ­irritant-induced airflow obstruction remain poorly understood. The non-selective cation channel Transient Receptor Potential Ankyrin 1 (TRPA1) has emerged as a promising target for therapeutic intervention in respiratory disorders as result of its unique biology that allows it to serve as the primary neuronal sensor not only for many of the hazardous irritants that cause chronic respiratory disease, but also for multiple inflammatory and oxidant mediators triggered by irritant exposure. Herein, we survey current evidence regarding TRPA1’s role as a sensor of an exceptionally wide array of oxidants, inflammatory mediators and hazardous air pollutants. Further, we review studies demonstrating that pharmacological inhibition or genetic disruption of TRPA1 protects laboratory animals from irritant-­triggered expulsion/dilution reflexes. Lastly, we conclude with a comprehensive overview of patented TRPA1 antagonists and highlight recent progress made toward the goal of developing TRPA1-targeted therapeutics.

TRPA1 Antagonists Block the Noxious Effects of Toxic Industrial Isocyanates and Tear Gases

The release of the methyl isocyanate in Bhopal, India, caused the worst industrial accident in history. Exposures to industrial isocyanates induce lacrimation, pain, airway irritation and edema. Similar responses are elicited by chemicals used as tear gases. Despite of frequent exposures the biological targets of isocyanates and tear gases in vivo have not been identified, precluding the development of effective countermeasures. We use Ca2+ imaging and electrophysiology to show that the noxious effects of isocyanates and all major tear gas agents are caused by activation of Ca2+ influx and membrane currents in mustard oil‐sensitive sensory neurons. These responses are mediated by Transient Receptor Potential Ankyrin 1 (TRPA1), an ion channel serving as a detector for reactive chemicals. In mice, genetic ablation or pharmacological inhibition of TRPA1 dramatically reduces isocyanate‐ and tear gas‐induced nocifensive behavior following both ocular and cutaneous exposures. We conclude that isocyanates and tear gas agents target the same neuronal receptor, TRPA1. TRPA1 antagonists can prevent and alleviate the adverse health effects of exposures to a wide range of toxic noxious chemicals.

Transient receptor potential ankyrin 1 antagonists block the noxious effects of toxic industrial isocyanates and tear gases

The release of methyl isocyanate in Bhopal, India, caused the worst industrial accident in history. Exposures to industrial isocyanates induce lacrimation, pain, airway irritation, and edema. Similar responses are elicited by chemicals used as tear gases. Despite frequent exposures, the biological targets of isocyanates and tear gases in vivo have not been identified, precluding the development of effective countermeasures. We use Ca(2+) imaging and electrophysiology to show that the noxious effects of isocyanates and those of all major tear gas agents are caused by activation of Ca(2+) influx and membrane currents in mustard oil-sensitive sensory neurons. These responses are mediated by transient receptor potential ankyrin 1 (TRPA1), an ion channel serving as a detector for reactive chemicals. In mice, genetic ablation or pharmacological inhibition of TRPA1 dramatically reduces isocyanate- and tear gas-induced nocifensive behavior after both ocular and cutaneous exposures. We conclude that isocyanates and tear gas agents target the same neuronal receptor, TRPA1. Treatment with TRPA1 antagonists may prevent and alleviate chemical irritation of the eyes, skin, and airways and reduce the adverse health effects of exposures to a wide range of toxic noxious chemicals.