Mammalian Transient Receptor Potential TRPA1 Channels: From Structure to Disease.

The transient receptor potential ankyrin (TRPA) channels are Ca2+-permeable nonselective cation channels remarkably conserved through the animal kingdom. Mammals have only one member, TRPA1, which is widely expressed in sensory neurons and in non-neuronal cells (such as epithelial cells and hair cells). TRPA1 owes its name to the presence of 14 ankyrin repeats located in the NH2 terminus of the channel, an unusual structural feature that may be relevant to its interactions with intracellular components. TRPA1 is primarily involved in the detection of an extremely wide variety of exogenous stimuli that may produce cellular damage. This includes a plethora of electrophilic compounds that interact with nucleophilic amino acid residues in the channel and many other chemically unrelated compounds whose only common feature seems to be their ability to partition in the plasma membrane. TRPA1 has been reported to be activated by cold, heat, and mechanical stimuli, and its function is modulated by multiple factors, including Ca2+, trace metals, pH, and reactive oxygen, nitrogen, and carbonyl species. TRPA1 is involved in acute and chronic pain as well as inflammation, plays key roles in the pathophysiology of nearly all organ systems, and is an attractive target for the treatment of related diseases. Here we review the current knowledge about the mammalian TRPA1 channel, linking its unique structure, widely tuned sensory properties, and complex regulation to its roles in multiple pathophysiological conditions.

Local immune response to food antigens drives meal-induced abdominal pain.

Up to 20% of people worldwide develop gastrointestinal symptoms following a meal1, leading to decreased quality of life, substantial morbidity and high medical costs. Although the interest of both the scientific and lay communities in this issue has increased markedly in recent years, with the worldwide introduction of gluten-free and other diets, the underlying mechanisms of food-induced abdominal complaints remain largely unknown. Here we show that a bacterial infection and bacterial toxins can trigger an immune response that leads to the production of dietary-antigen-specific IgE antibodies in mice, which are limited to the intestine. Following subsequent oral ingestion of the respective dietary antigen, an IgE- and mast-cell-dependent mechanism induced increased visceral pain. This aberrant pain signalling resulted from histamine receptor H1-mediated sensitization of visceral afferents. Moreover, injection of food antigens (gluten, wheat, soy and milk) into the rectosigmoid mucosa of patients with irritable bowel syndrome induced local oedema and mast cell activation. Our results identify and characterize a peripheral mechanism that underlies food-induced abdominal pain, thereby creating new possibilities for the treatment of irritable bowel syndrome and related abdominal pain disorders.

YF17D-based vaccines - standing on the shoulders of a giant.

Live-attenuated yellow fever vaccine (YF17D) was developed in the 1930s as the first ever empirically derived human vaccine. Ninety years later, it is still a benchmark for vaccines made today. YF17D triggers a particularly broad and polyfunctional response engaging multiple arms of innate, humoral and cellular immunity. This unique immunogenicity translates into an extraordinary vaccine efficacy and outstanding longevity of protection, possibly by single-dose immunization. More recently, progress in molecular virology and synthetic biology allowed engineering of YF17D as a powerful vector and promising platform for the development of novel recombinant live vaccines, including two licensed vaccines against Japanese encephalitis and dengue, even in paediatric use. Likewise, numerous chimeric and transgenic preclinical candidates have been described. These include prophylactic vaccines against emerging viral infections (e.g. Lassa, Zika and SARS-CoV-2) and parasitic diseases (e.g. malaria), as well as therapeutic applications targeting persistent infections (e.g. HIV and chronic hepatitis), and cancer. Efforts to overcome historical safety concerns and manufacturing challenges are ongoing and pave the way for wider use of YF17D-based vaccines. In this review, we summarize recent insights regarding YF17D as vaccine platform, and how YF17D-based vaccines may complement as well as differentiate from other emerging modalities in response to unmet medical needs and for pandemic preparedness.

Extremely Low-Frequency Electromagnetic Stimulation (ELF-EMS) Improves Neurological Outcome and Reduces Microglial Reactivity in a Rodent Model of Global Transient Stroke.

Extremely low-frequency electromagnetic stimulation (ELF-EMS) was demonstrated to be significantly beneficial in rodent models of permanent stroke. The mechanism involved enhanced cerebrovascular perfusion and endothelial cell nitric oxide production. However, the possible effect on the neuroinflammatory response and its efficacy in reperfusion stroke models remains unclear. To evaluate ELF-EMS effectiveness and possible immunomodulatory response, we studied neurological outcome, behavior, neuronal survival, and glial reactivity in a rodent model of global transient stroke treated with 13.5 mT/60 Hz. Next, we studied microglial cells migration and, in organotypic hippocampal brain slices, we assessed neuronal survival and microglia reactivity. ELF-EMS improved the neurological score and behavior in the ischemia-reperfusion model. It also improved neuronal survival and decreased glia reactivity in the hippocampus, with microglia showing the first signs of treatment effect. In vitro ELF-EMS decreased (Lipopolysaccharide) LPS and ATP-induced microglia migration in both scratch and transwell assay. Additionally, in hippocampal brain slices, reduced microglial reactivity, improved neuronal survival, and modulation of inflammation-related markers was observed. Our study is the first to show that an EMF treatment has a direct impact on microglial migration. Furthermore, ELF-EMS has beneficial effects in an ischemia/reperfusion model, which indicates that this treatment has clinical potential as a new treatment against ischemic stroke.

Acute inhibition of transient receptor potential vanilloid-type 4 cation channel halts cytoskeletal dynamism in microglia.

Microglia, the resident macrophages of the central nervous system, are highly motile cells that support brain development, provision neuronal signaling, and protect brain cells against damage. Proper microglial functioning requires constant cell movement and morphological changes. Interestingly, the transient receptor potential vanilloid 4 (TRPV4) channel, a calcium-permeable channel, is involved in hypoosmotic morphological changes of retinal microglia and regulates temperature-dependent movement of microglial cells both in vitro and in vivo. Despite the broad functions of TRPV4 and the recent findings stating a role for TRPV4 in microglial movement, little is known about how TRPV4 modulates cytoskeletal remodeling to promote changes of microglial motility. Here we show that acute inhibition of TRPV4, but not its constitutive absence in the Trpv4 KO cells, affects the morphology and motility of microglia in vitro. Using high-end confocal imaging techniques, we show a decrease in actin-rich filopodia and tubulin dynamics upon acute inhibition of TRPV4 in vitro. Furthermore, using acute brain slices we demonstrate that Trpv4 knockout microglia display lower ramification complexity, slower process extension speed and consequently smaller surveyed area. We conclude that TRPV4 inhibition triggers a shift in cytoskeleton remodeling of microglia influencing their migration and morphology.

p27kip1 Modulates the Morphology and Phagocytic Activity of Microglia.

p27kip1 is a multifunctional protein that promotes cell cycle exit by blocking the activity of cyclin/cyclin-dependent kinase complexes as well as migration and motility via signaling pathways that converge on the actin and microtubule cytoskeleton. Despite the broad characterization of p27kip1 function in neural cells, little is known about its relevance in microglia. Here, we studied the role of p27kip1 in microglia using a combination of in vitro and in situ approaches. While the loss of p27kip1 did not affect microglial density in the cerebral cortex, it altered their morphological complexity in situ. However, despite the presence of p27kip1 in microglial processes, as shown by immunofluorescence in cultured cells, loss of p27kip1 did not change microglial process motility and extension after applying laser-induced brain damage in cortical brain slices. Primary microglia lacking p27kip1 showed increased phagocytic uptake of synaptosomes, while a cell cycle dead variant negatively affected phagocytosis. These findings indicate that p27kip1 plays specific roles in microglia.

Effect of resolvins on sensitisation of TRPV1 and visceral hypersensitivity in IBS.

OBJECTIVE: Resolvins (RvD1, RvD2 and RvE1) are endogenous anti-inflammatory lipid mediators that display potent analgesic properties in somatic pain by modulating transient receptor potential vanilloid 1 (TRPV1) activation. To what extent these molecules could also have a beneficial effect on TRPV1 sensitisation and visceral hypersensitivity (VHS), mechanisms involved in IBS, remains unknown. DESIGN: The effect of RvD1, RvD2 and RvE1 on TRPV1 activation and sensitisation by histamine or IBS supernatants was assessed on murine dorsal root ganglion (DRG) neurons using live Ca2+ imaging. Based on the results obtained in vitro, we further studied the effect of RvD2 in vivo using a murine model of post-infectious IBS and a rat model of post-inflammatory VHS. Finally, we also tested the effect of RvD2 on submucosal neurons in rectal biopsies of patients with IBS. RESULTS: RvD1, RvD2 and RvE1 prevented histamine-induced TRPV1 sensitisation in DRG neurons at doses devoid of an analgesic effect. Of note, RvD2 also reversed TRPV1 sensitisation by histamine and IBS supernatant. This effect was blocked by the G protein receptor 18 (GPR18) antagonist O-1918 (3-30 µM) and by pertussis toxin. In addition, RvD2 reduced the capsaicin-induced Ca2+ response of rectal submucosal neurons of patients with IBS. Finally, treatment with RvD2 normalised pain responses to colorectal distention in both preclinical models of VHS. CONCLUSIONS: Our data suggest that RvD2 and GPR18 agonists may represent interesting novel compounds to be further evaluated as treatment for IBS.

Staphylococcus aureus enterotoxin B disrupts nasal epithelial barrier integrity.

BACKGROUND: Staphylococcus aureus colonization and release of enterotoxin B (SEB) has been associated with severe chronic rhinosinusitis with nasal polyps (CRSwNP). The pathogenic mechanism of SEB on epithelial barriers, however, is largely unexplored. OBJECTIVE: We investigated the effect of SEB on nasal epithelial barrier function. METHODS: SEB was apically administered to air-liquid interface (ALI) cultures of primary polyp and nasal epithelial cells of CRSwNP patients and healthy controls, respectively. Epithelial cell integrity and tight junction expression were evaluated. The involvement of Toll-like receptor 2 (TLR2) activation was studied in vitro with TLR2 monoclonal antibodies and in vivo in tlr2-/- knockout mice. RESULTS: SEB applied to ALI cultures of polyp epithelial cells decreased epithelial cell integrity by diminishing occludin and zonula occludens (ZO)-1 protein expression. Antagonizing TLR2 prevented SEB-induced barrier disruption. SEB applied in the nose of control mice increased mucosal permeability and decreased mRNA expression of occludin and ZO-1, whereas mucosal integrity and tight junction expression remained unaltered in tlr2-/- mice. Furthermore, in vitro SEB stimulation resulted in epithelial production of IL-6 and IL-8, which was prevented by TLR2 antagonization. CONCLUSION & CLINICAL RELEVANCE: SEB damages nasal polyp epithelial cell integrity by triggering TLR2 in CRSwNP. Our results suggest that SEB might represent a driving factor of disease exacerbation, rather than a causal factor for epithelial defects in CRSwNP. Interfering with TLR2 triggering might provide a way to avoid the pathophysiological consequences of S. aureus on inflammation in CRSwNP.

The Agonist Action of Alkylphenols on TRPA1 Relates to Their Effects on Membrane Lipid Order: Implications for TRPA1-Mediated Chemosensation.

The Transient Receptor Potential Ankyrin 1 cation channel (TRPA1) is a broadly-tuned chemosensor expressed in nociceptive neurons. Multiple TRPA1 agonists are chemically unrelated non-electrophilic compounds, for which the mechanisms of channel activation remain unknown. Here, we assess the hypothesis that such chemicals activate TRPA1 by inducing mechanical perturbations in the plasma membrane. We characterized the activation of mouse TRPA1 by non-electrophilic alkylphenols (APs) of different carbon chain lengths in the para position of the aromatic ring. Having discarded oxidative stress and the action of electrophilic mediators as activation mechanisms, we determined whether APs induce mechanical perturbations in the plasma membrane using dyes whose fluorescence properties change upon alteration of the lipid environment. APs activated TRPA1, with potency increasing with their lipophilicity. APs increased the generalized polarization of Laurdan fluorescence and the anisotropy of the fluorescence of 1,6-diphenyl-1,3,5-hexatriene (DPH), also according to their lipophilicity. Thus, the potency of APs for TRPA1 activation is an increasing function of their ability to induce lipid order and membrane rigidity. These results support the hypothesis that TRPA1 senses non-electrophilic compounds by detecting the mechanical alterations they produce in the plasma membrane. This may explain how structurally unrelated non-reactive compounds induce TRPA1 activation and support the role of TRPA1 as an unspecific sensor of potentially noxious compounds.

Preoperative administration of the 5-HT4 receptor agonist prucalopride reduces intestinal inflammation and shortens postoperative ileus via cholinergic enteric neurons.

OBJECTIVES: Vagus nerve stimulation (VNS), most likely via enteric neurons, prevents postoperative ileus (POI) by reducing activation of alpha7 nicotinic receptor (α7nAChR) positive muscularis macrophages (mMφ) and dampening surgery-induced intestinal inflammation. Here, we evaluated if 5-HT4 receptor (5-HT4R) agonist prucalopride can mimic this effect in mice and human. DESIGN: Using Ca2+ imaging, the effect of electrical field stimulation (EFS) and prucalopride was evaluated in situ on mMφ activation evoked by ATP in jejunal muscularis tissue. Next, preoperative and postoperative administration of prucalopride (1-5 mg/kg) was compared with that of preoperative VNS in a model of POI in wild-type and α7nAChR knockout mice. Finally, in a pilot study, patients undergoing a Whipple procedure were preoperatively treated with prucalopride (n=10), abdominal VNS (n=10) or sham/placebo (n=10) to evaluate the effect on intestinal inflammation and clinical recovery of POI. RESULTS: EFS reduced the ATP-induced Ca2+ response of mMφ, an effect that was dampened by neurotoxins tetrodotoxin and ω-conotoxin and mimicked by prucalopride. In vivo, prucalopride administered before, but not after abdominal surgery reduced intestinal inflammation and prevented POI in wild-type, but not in α7nAChR knockout mice. In humans, preoperative administration of prucalopride, but not of VNS, decreased Il6 and Il8 expression in the muscularis externa and improved clinical recovery. CONCLUSION: Enteric neurons dampen mMφ activation, an effect mimicked by prucalopride. Preoperative, but not postoperative treatment with prucalopride prevents intestinal inflammation and shortens POI in both mice and human, indicating that preoperative administration of 5-HT4R agonists should be further evaluated as a treatment of POI. TRIAL REGISTRATION NUMBER: NCT02425774.

Activation of the cation channel TRPM3 in perivascular nerves induces vasodilation of resistance arteries.

The Transient Receptor Potential Melastatin 3 (TRPM3) is a Ca2+-permeable non-selective cation channel activated by the neurosteroid pregnenolone sulfate (PS). This compound was previously shown to contract mouse aorta by activating TRPM3 in vascular smooth muscle cells (VSMC), and proposed as therapeutic modulator of vascular functions. However, PS effects and the role of TRPM3 in resistance arteries remain unknown. Thus, we aimed at determining the localization and physiological role of TRPM3 in mouse mesenteric arteries. Real-time qPCR experiments, anatomical localization using immunofluorescence microscopy and patch-clamp recordings in isolated VSMC showed that TRPM3 expression in mesenteric arteries is restricted to perivascular nerves. Pressure myography experiments in wild type (WT) mouse arteries showed that PS vasodilates with a concentration-dependence that was best fit by two Hill components (effective concentrations, EC50, of 14 and 100 µM). The low EC50 component was absent in preparations from Trpm3 knockout (KO) mice and in WT arteries in the presence of the CGRP receptor antagonist BIBN 4096. TRPM3-dependent vasodilation was partially inhibited by a cocktail of K+ channel blockers, and not mediated by ß-adrenergic signaling. We conclude that, contrary to what was found in aorta, PS dilates mesenteric arteries, partly via an activation of TRPM3 that triggers CGRP release from perivascular nerve endings and a subsequent activation of K+ channels in VSMC. We propose that TRPM3 is implicated in the regulation of the tone of resistance arteries and that its activation by yet unidentified endogenous damage-associated molecules lead to protective vasodilation responses in mesenteric arteries.

Expression and Functional Role of TRPV4 in Bone Marrow-Derived CD11c+ Cells.

The increase in cytosolic Ca2+ is essential in key effector functions of dendritic cells (DCs), including differentiation, maturation, cytokine expression, and phagocytosis. Although several Ca2+-permeable ion channels have been described in DCs, the contribution of transient receptor potential (TRP) channels remains poorly understood. Here, we investigated whether TRPV4 plays a role in the differentiation, maturation, and phagocytosis of granulocyte-macrophage colony-stimulating factor (GM-CSF)-induced mouse bone marrow-derived cells (BMDCs). Using intracellular Ca2+ imaging experiments, we found that TRPV4 was functionally expressed in the plasma membrane of immature CD11c+ BMDCs and that its activity and expression were downregulated in CD11c+ BMDCs matured with lipopolysaccharide (LPS). Comparative analysis of the GM-CSF-stimulated cells showed that Trpv4 knockout and wild-type bone marrow cultures had a similar distribution of differentiated cells, generating a heterogenous culture population rich in CD11c+, CD11b+ cells, and low levels of F4/80+ cells. The lack of TRPV4 did not prevent the LPS-induced nuclear translocation of NF-κB, the upregulation of the proinflammatory cytokines IL-6 and IL-12, or the upregulation of the maturation markers CD40, CD80, and CD86. In contrast, TRPV4-deficient CD11c+ BMDCs exhibited a significantly reduced endocytic capacity of IgG-coated beads, but the internalization of uncoated beads in the absence of TRPV4 was not affected. Taken together, our results demonstrate that TRPV4 was dispensable in the differentiation and maturation of mouse CD11c+ BMDCs but contributed to the mechanism underlying Fc receptor-mediated phagocytosis. Overall, our results further strengthen the role of TRPV4 in immune-related processes.

Enhanced chemosensory sensitivity in patients with idiopathic rhinitis and its reversal by nasal capsaicin treatment.

BACKGROUND: The therapeutic action of capsaicin treatment in patients with idiopathic rhinitis (IR) is based on ablation of the transient receptor potential cation channel subfamily V, receptor 1 (TRPV1)-substance P nociceptive signaling pathway. However, the functional consequences of capsaicin treatment on nasal nerve activation and the association between the reduction in nasal hyperreactivity (NHR) and response to capsaicin treatment remain unknown. OBJECTIVE: We sought to study the effects of capsaicin nasal spray on the afferent innervation of the nasal mucosa by monitoring trigeminal nerve activity in patients with IR and healthy control (HC) subjects. METHODS: A double-blind, placebo-controlled randomized trial with capsaicin nasal spray was performed involving 33 patients with IR and 12 HC subjects. Before and at 4, 12, and 26 weeks after treatment, nasal mucosal potentials (NMPs) were measured while exposing the nasal mucosa of patients with IR and HC subjects to aerosols with increasing doses of the chemical irritants allyl isothiocyanate (AITC; also known as mustard oil) or capsaicin. The threshold for each compound was determined for each subject. The results of the NMP measurements were evaluated in parallel with the therapeutic response, visual analog scale scores for nasal symptoms, self-reported NHR, and mRNA expression of PGP9.5; TRPV1; transient receptor potential cation channel subfamily A, receptor 1 (TRPA1); TRPV4; transient receptor potential cation channel subfamily M, member 8 (TRPM8); and nerve growth factor (NGF) in nasal biopsy specimens. RESULTS: AITC turned out to be the best stimulus because the coughing induced by capsaicin interfered with measurements. At baseline, the threshold for evoking changes in NMPs based on AITC was significantly lower for patients with IR compared with HC subjects (P = .0423). Capsaicin treatment of IR patients increased the threshold for the response to AITC at 4 and 12 weeks compared with placebo (P = .0406 and P = .0325, respectively), which returned to baseline by week 26 (P = .0611). This increase correlated with changes in visual analog scale major symptom (P = .0004) and total symptom (P = .0018) scores. IR patients with self-reported NHR at baseline showed a trend to being better responders to capsaicin treatment compared with patients with IR but without NHR (P = .10). CONCLUSION: The lower threshold for AITC based on NMPs in patients with IR compared with HC subjects and the increased threshold for AITC after capsaicin treatment in patients with IR demonstrate the crucial role of TRPA1 and TRPV1 in IR pathophysiology. The strong correlation between the increase in AITC threshold in patients with IR and symptom reduction after capsaicin treatment demonstrates the clinical relevance of these findings.

Histamine Receptor H1-Mediated Sensitization of TRPV1 Mediates Visceral Hypersensitivity and Symptoms in Patients With Irritable Bowel Syndrome.

BACKGROUND & AIMS: Histamine sensitizes the nociceptor transient reporter potential channel V1 (TRPV1) and has been shown to contribute to visceral hypersensitivity in animals. We investigated the role of TRPV1 in irritable bowel syndrome (IBS) and evaluated if an antagonist of histamine receptor H1 (HRH1) could reduce symptoms of patients in a randomized placebo-controlled trial. METHODS: By using live calcium imaging, we compared activation of submucosal neurons by the TRPV1 agonist capsaicin in rectal biopsy specimens collected from 9 patients with IBS (ROME 3 criteria) and 15 healthy subjects. The sensitization of TRPV1 by histamine, its metabolite imidazole acetaldehyde, and supernatants from biopsy specimens was assessed by calcium imaging of mouse dorsal root ganglion neurons. We then performed a double-blind trial of patients with IBS (mean age, 31 y; range, 18-65 y; 34 female). After a 2-week run-in period, subjects were assigned randomly to groups given either the HRH1 antagonist ebastine (20 mg/day; n = 28) or placebo (n = 27) for 12 weeks. Rectal biopsy specimens were collected, barostat studies were performed, and symptoms were assessed (using the validated gastrointestinal symptom rating scale) before and after the 12-week period. Patients were followed up for an additional 2 weeks. Abdominal pain, symptom relief, and health-related quality of life were assessed on a weekly basis. The primary end point of the study was the effect of ebastine on the symptom score evoked by rectal distension. RESULTS: TRPV1 responses of submucosal neurons from patients with IBS were potentiated compared with those of healthy volunteers. Moreover, TRPV1 responses of submucosal neurons from healthy volunteers could be potentiated by their pre-incubation with histamine; this effect was blocked by the HRH1 antagonist pyrilamine. Supernatants from rectal biopsy specimens from patients with IBS, but not from the healthy volunteers, sensitized TRPV1 in mouse nociceptive dorsal root ganglion neurons via HRH1; this effect could be reproduced by histamine and imidazole acetaldehyde. Compared with subjects given placebo, those given ebastine had reduced visceral hypersensitivity, increased symptom relief (ebastine 46% vs placebo 13%; P = .024), and reduced abdominal pain scores (ebastine 39 ± 23 vs placebo 62 ± 22; P = .0004). CONCLUSIONS: In studies of rectal biopsy specimens from patients, we found that HRH1-mediated sensitization of TRPV1 is involved in IBS. Ebastine, an antagonist of HRH1, reduced visceral hypersensitivity, symptoms, and abdominal pain in patients with IBS. Inhibitors of this pathway might be developed as a new treatment approach for IBS. ClinicalTrials.gov no: NCT01144832.

Silica nanoparticles inhibit the cation channel TRPV4 in airway epithelial cells.

BACKGROUND: Silica nanoparticles (SiNPs) have numerous beneficial properties and are extensively used in cosmetics and food industries as anti-caking, densifying and hydrophobic agents. However, the increasing exposure levels experienced by the general population and the ability of SiNPs to penetrate cells and tissues have raised concerns about possible toxic effects of this material. Although SiNPs are known to affect the function of the airway epithelium, the molecular targets of these particles remain largely unknown. Given that SiNPs interact with the plasma membrane of epithelial cells we hypothesized that they may affect the function of Transient Receptor Potential Vanilloid 4 (TRPV4), a cation-permeable channel that regulates epithelial barrier function. The main aims of this study were to evaluate the effects of SiNPs on the activation of TRPV4 and to determine whether these alter the positive modulatory action of this channel on the ciliary beat frequency in airway epithelial cells. RESULTS: Using fluorometric measurements of intracellular Ca2+ concentration ([Ca2+]i) we found that SiNPs inhibit activation of TRPV4 by the synthetic agonist GSK1016790A in cultured human airway epithelial cells 16HBE and in primary cultured mouse tracheobronchial epithelial cells. Inhibition of TRPV4 by SiNPs was confirmed in intracellular Ca2+ imaging and whole-cell patch-clamp experiments performed in HEK293T cells over-expressing this channel. In addition to these effects, SiNPs were found to induce a significant increase in basal [Ca2+]i, but in a TRPV4-independent manner. SiNPs enhanced the activation of the capsaicin receptor TRPV1, demonstrating that these particles have a specific inhibitory action on TRPV4 activation. Finally, we found that SiNPs abrogate the increase in ciliary beat frequency induced by TRPV4 activation in mouse airway epithelial cells. CONCLUSIONS: Our results show that SiNPs inhibit TRPV4 activation, and that this effect may impair the positive modulatory action of the stimulation of this channel on the ciliary function in airway epithelial cells. These findings unveil the cation channel TRPV4 as a primary molecular target of SiNPs.

Different ligands of the TRPV3 cation channel cause distinct conformational changes as revealed by intrinsic tryptophan fluorescence quenching.

TRPV3 is a thermosensitive ion channel primarily expressed in epithelial tissues of the skin, nose, and tongue. The channel has been implicated in environmental thermosensation, hyperalgesia in inflamed tissues, skin sensitization, and hair growth. Although transient receptor potential (TRP) channel research has vastly increased our understanding of the physiological mechanisms of nociception and thermosensation, the molecular mechanics of these ion channels are still largely elusive. In order to better comprehend the functional properties and the mechanism of action in TRP channels, high-resolution three-dimensional structures are indispensable, because they will yield the necessary insights into architectural intimacies at the atomic level. However, structural studies of membrane proteins are currently hampered by difficulties in protein purification and in establishing suitable crystallization conditions. In this report, we present a novel protocol for the purification of membrane proteins, which takes advantage of a C-terminal GFP fusion. Using this protocol, we purified human TRPV3. We show that the purified protein is a fully functional ion channel with properties akin to the native channel using planar patch clamp on reconstituted channels and intrinsic tryptophan fluorescence spectroscopy. Using intrinsic tryptophan fluorescence spectroscopy, we reveal clear distinctions in the molecular interaction of different ligands with the channel. Altogether, this study provides powerful tools to broaden our understanding of ligand interaction with TRPV channels, and the availability of purified human TRPV3 opens up perspectives for further structural and functional studies.

Neuro-immune interactions in chemical-induced airway hyperreactivity.

Asthma may be induced by chemical sensitisers, via mechanisms that are still poorly understood. This type of asthma is characterised by airway hyperreactivity (AHR) and little airway inflammation. Since potent chemical sensitisers, such as toluene-2,4-diisocyanate (TDI), are also sensory irritants, it is suggested that chemical-induced asthma relies on neuro-immune mechanisms.We investigated the involvement of transient receptor potential channels (TRP) A1 and V1, major chemosensors in the airways, and mast cells, known for their ability to communicate with sensory nerves, in chemical-induced AHR.In vitro intracellular calcium imaging and patch-clamp recordings in TRPA1- and TRPV1-expressing Chinese hamster ovarian cells showed that TDI activates murine TRPA1, but not TRPV1. Using an in vivo model, in which an airway challenge with TDI induces AHR in TDI-sensitised C57Bl/6 mice, we demonstrated that AHR does not develop, despite successful sensitisation, in Trpa1 and Trpv1 knockout mice, and wild-type mice pretreated with a TRPA1 blocker or a substance P receptor antagonist. TDI-induced AHR was also abolished in mast cell deficient Kit(Wsh) (/Wsh) mice, and in wild-type mice pretreated with the mast cell stabiliser ketotifen, without changes in immunological parameters.These data demonstrate that TRPA1, TRPV1 and mast cells play an indispensable role in the development of TDI-elicited AHR.

Capsaicin treatment reduces nasal hyperreactivity and transient receptor potential cation channel subfamily V, receptor 1 (TRPV1) overexpression in patients with idiopathic rhinitis.

BACKGROUND: Idiopathic rhinitis (IR) is a prevalent condition for which capsaicin nasal spray is the most effective treatment. However, the mechanisms underlying IR and the therapeutic action of capsaicin remain unknown. OBJECTIVE: We sought to investigate the molecular and cellular bases of IR and the therapeutic action of capsaicin. METHODS: Fourteen patients with IR and 12 healthy control subjects (HCs) were treated with intranasal capsaicin. The therapeutic effect was assessed in patients with IR by using visual analog scale and therapeutic response evaluation scores, and nasal hyperreactivity was evaluated by means of cold dry air provocation. Nasal samples served to measure the levels of neuromediators and expression of chemosensory cation channels, protein gene product 9.5 (PGP 9.5), and the mast cell marker c-kit. The effects of capsaicin were also tested in vitro on human nasal epithelial cells and mast cells. RESULTS: Patients with IR had higher baseline transient receptor potential cation channel subfamily V, receptor 1 (TRPV1) expression in the nasal mucosa and higher concentrations of substance P (SP) in nasal secretions than HCs. Symptomatic relief was observed in 11 of 14 patients with IR after capsaicin treatment. Expression of TRPV1; transient receptor potential cation channel subfamily M, receptor 8 (TRPM8); and PGP 9.5 was only reduced in patients with IR after capsaicin treatment. Capsaicin did not alter c-KIT expression or nasal epithelial morphology in patients with IR and HCs nor did it induce apoptosis or necrosis in cultured human nasal epithelial cells and mast cells. CONCLUSION: IR features an overexpression of TRPV1 in the nasal mucosa and increased SP levels in nasal secretions. Capsaicin exerts its therapeutic action by ablating the TRPV1-SP nociceptive signaling pathway in the nasal mucosa.

Allyl isothiocyanate sensitizes TRPV1 to heat stimulation.

The powerful plant-derived irritant allyl isothiocyanate (AITC, aka mustard oil) induces hyperalgesia to heat in rodents and humans through mechanisms that are not yet fully understood. It is generally believed that AITC activates the broadly tuned chemosensory cation channel transient receptor potential cation channel subfamily A member 1 (TRPA1), triggering an inflammatory response that sensitizes the heat sensor transient receptor potential cation channel subfamily V member 1 (TRPV1). In the view of recent data demonstrating that AITC can directly activate TRPV1, we here explored the possibility that this compound sensitizes TRPV1 to heat stimulation in a TRPA1-independent manner. Patch-clamp recordings and intracellular Ca(2+) imaging experiments in HEK293T cells over-expressing mouse TRPV1 revealed that the increase in channel activation induced by heating is larger in the presence of AITC than in control conditions. The analysis of the effects of AITC and heat on the current-voltage relationship of TRPV1 indicates that the mechanism of sensitization is based on additive shifts of the voltage dependence of activation towards negative voltages. Finally, intracellular Ca(2+) imaging experiments in mouse sensory neurons isolated from Trpa1 KO mice yielded that AITC enhances the response to heat, specifically in the subpopulation expressing TRPV1. Furthermore, this effect was strongly reduced by the TRPV1 inhibitor capsazepine and virtually absent in neurons isolated from double Trpa1/Trpv1 KO mice. Taken together, these findings demonstrate that TRPV1 is a locus for cross sensitization between AITC and heat in sensory neurons and may help explaining, at least in part, the role of this channel in AITC-induced hyperalgesia to heat.

Cinnamaldehyde inhibits L-type calcium channels in mouse ventricular cardiomyocytes and vascular smooth muscle cells.

Cinnamaldehyde (CA), a major component of cinnamon, is known to have important actions in the cardiovascular system, including vasorelaxation and decrease in blood pressure. Although CA-induced activation of the chemosensory cation channel TRPA1 seems to be involved in these phenomena, it has been shown that genetic ablation of Trpa1 is insufficient to abolish CA effects. Here, we confirm that CA relaxes rat aortic rings and report that it has negative inotropic and chronotropic effects on isolated mouse hearts. Considering the major role of L-type Ca(2+) channels in the control of the vascular tone and cardiac contraction, we used whole-cell patch-clamp to test whether CA affects L-type Ca(2+) currents in mouse ventricular cardiomyocytes (VCM, with Ca(2+) as charge carrier) and in mesenteric artery smooth muscle cells (VSMC, with Ba(2+) as charge carrier). We found that CA inhibited L-type currents in both cell types in a concentration-dependent manner, with little voltage-dependent effects. However, CA was more potent in VCM than in VSMC and caused opposite effects on the rate of inactivation. We found these divergences to be at least in part due to the use of different charge carriers. We conclude that CA inhibits L-type Ca(2+) channels and that this effect may contribute to its vasorelaxing action. Importantly, our results demonstrate that TRPA1 is not a specific target of CA and indicate that the inhibition of voltage-gated Ca(2+) channels should be taken into account when using CA to probe the pathophysiological roles of TRPA1.