TRPA1 Channels Research Articles

Potential Effects of Melatonin on TRPA1 Channels in the Prevention and Treatment of Alzheimer's Disease.

Alzheimer's disease (AD) is the most common cause of dementia and is defined as a progressive neurodegenerative disease. Main clinical features of AD are progressive impairment in learning and memory loss. Several studies have indicated that mitochondria play a critical role in the pathogenesis of AD. In this study, we investigated the effect of melatonin on mitochondria-dependent TRPA1 ion channels in neuroblastoma cells by creating an in vitro model of Alzheimer's disease. Okadaic acid was applied to SH-SY5Y (human neuroblastoma cell line) cells to create an AD model. After cellular differentiation, the following 7 main groups were created: Group 1 (Control), Group 2 (Mel+AD), Group 3 (Mel+AD+AP18), Group 4 (AD), Group 5 (AD+AP18), Group 6 (AD+Mel), and Group 7 (AD+Mel+AP18), and Alzheimer's disease was determined in vitro by examining the effect of melatonin on calcium-dependent TRPA1 channels in neuroblastoma cells. The Ca2+concentration was greater in the melatonin+AD, AD and AD+melatonin groups than in the control (p<0.001). However, there was no statistically significant difference between Mel+AD+AP18, AD+Mel+AP18 and the control. We determined that Ca2+ levels were lower in the melatonin+AD and AD+melatonin groups than in the AD group (p<0.001 and p<0.05). Additionally, cytosolic Ca2+ concentrations were found to be lower in the melatonin+AD group than in the AD+melatonin group (p<0.05). In evaluating the apoptosis and oxidative stress levels, we found that the apoptosis and intracellular ROS values were higher in the melatonin+AD, AD and AD+melatonin groups than in the control (p<0.001). In this respect, the mitochondrial depolarization and caspase-3 and caspase-9 levels were higher in the melatonin+AD, AD and AD+melatonin groups than in the control group (p<0.001). Additionally, the mitochondrial depolarization, caspase-3 and caspase-9 values were higher in the AD group than in the melatonin+AD and AD+melatonin groups (p<0.001), while mitochondrial depolarization and caspase-3 levels were lower in the melatonin+AD group than in the AD+melatonin group (p<0.001). However, in the same groups, there was no statistically significant difference in caspase-9 results. Additionally, the caspase-9 values were lower in the melatonin+AD group, AD group and AD+melatonin groups than in the melatonin+AD+AP18, AD+AP18 and AD+melatonin+AP18 groups, respectively (p<0.001 and p<0.05). Our results suggest that melatonin may be an effective option in the treatment and prophylaxis of Alzheimer's disease by reducing cytosolic Ca2+ concentration, apoptosis and intracellular ROS through TRPA1 channels.

Agonistic/antagonistic properties of lactones in food flavors on the sensory ion channels TRPV1 and TRPA1.

Flavor compounds provide aroma and sensations in the oral cavity. They are not present alone in the oral cavity, but rather in combination with several other food ingredients. This study aimed to clarify the relationship between the mixing of pungent flavor compounds and the response of pungent receptors, TRPV1 and TRPA1 channels. We focused on lactones that activate TRPV1 despite their presence in bland foods, such as dairy products and fruits, and analyzed their interaction with receptors using TRPV1- and TRPA1-expressing HEK293 cells. We found that γ-octalactone, γ-nonalactone, and δ-nonalactone activated TRPA1. When mixed with pungent components, some γ- and δ-lactones inhibited capsaicin-mediated TRPV1 responses, and δ-dodecalactone inhibited allyl isothiocyanate-mediated TRPA1 responses. Furthermore, the dose-response relationship of capsaicin and γ-nonalactone to TRPV1 suggests that γ-nonalactone acts as an agonist or antagonist of TRPV1, depending on its concentration. Conversely, γ-nonalactone and δ-dodecalactone were found to act only as agonists and antagonists, respectively, against TRPA1. These results suggest that lactones in foods may not only endow food with aroma, but also play a role in modulating food pungency by acting on TRPV1 and TRPA1. The dose-response relationships of a mixture of flavor compounds with TRPV1 and TRPA1 provide insights into the molecular physiological basis of pungency that may be the cornerstone for developing new spice mix recipes.

A fluorogenic probe for TRPA1 channel imaging based on a molecular rotation mechanism

A fluorescent probe for selectively visualizing the TRPA1 channel and rapidly screening its regulators.

The astrocytic TRPA1 channel plays a protective role in vascular cognitive impairment

The astrocytic TRPA1 channel plays a protective role in vascular cognitive impairment

Expression and Metabolic Functions of the Thermally Activated Transient Receptor Potential Channels TRPA1 and TRPM8 in Human Myotubes

Expression and Metabolic Functions of the Thermally Activated Transient Receptor Potential Channels TRPA1 and TRPM8 in Human Myotubes

Selective activation of TRPA1 ion channels by nitrobenzene skin sensitizers DNFB and DNCB

2, 4-dinitrofluorobenzene (DNFB) and 2, 4-dinitrochlorobenzene (DNCB) are well known as skin sensitizers that can cause dermatitis. DNFB has shown to more potently sensitize skin; however, how DNFB and DNCB cause skin inflammation at a molecular level and why this difference in their sensitization ability is observed remain unknown. In this study, we aimed to identify the molecular targets and mechanisms on which DNFB and DNCB act. We used a fluorescent calcium imaging plate reader in an initial screening assay before patch-clamp recordings for validation. Molecular docking in combination with site-directed mutagenesis was then carried out to investigate DNFB and DNCB binding sites in the TRPA1 ion channel that may be selectively activated by these tow sensitizers. We found that DNFB and DNCB selectively activated TRPA1 channel with EC50 values of 2.3 ± 0.7 μM and 42.4 ± 20.9 μM, respectively. Single-channel recordings revealed that DNFB and DNCB increase the probability of channel opening and act on three residues (C621, E625, and Y658) critical for TRPA1 activation. Our findings may not only help explain the molecular mechanism underlying the dermatitis and pruritus caused by chemicals such as DNFB and DNCB, but also provide a molecular tool 7.5-fold more potent than the current TRPA1 activator allyl isothiocyanate (AITC) used for investigating TRPA1 channel pharmacology and pathology.

TRPA1-mediated effects on the functional activity of macrophages under the exposure with cigarette smoke and cinnamaldehyde

Introduction. Being the leading cause of COPD, smoking represents a major health problem. Upon entering the respiratory tract, cigarette smoke comes into contact with various cells, including macrophages expressing on their surface TRPA1 receptors, which are sensitive to the main pathogenic compounds formed during tobacco combustion.Aim. To study the functional activity of TRPA1 channels on macrophages in terms of cell responses to cigarette smoke and the TRPA1 agonist cinnamaldehyde (CA). Materials and methods. The experimental conditions included exposure of monocyte-derived macrophages to CA (100 μM), 4% cigarette smoke extract (CSE) and 4% CSE after pretreatment with TRPA1 selective antagonist (HC-030031 100 μM). The concentration of cytokines in the culture medium, the expression of TRPA1 on the cell surface, as well as the phagocytic activity of macrophages were analyzed by flow cytometry.Results. We found that 60.2 (49.6; 71.8)% of cells expressed TRPA1 and their number increased after exposure with CA. CSE significantly inhibited CXCL10 production from 1121.3 (295.7; 3154.6) pg/ml to 187.9 (113.8; 398.3) pg/ml (p=0.04), which was partially prevented by blocking TRPA1 (692.4 [428.6; 2916.6] pg/ml, p=0.04). Similar to CSE, CA also caused a decrease in CXCL10 concentration (189.2 [111.7; 311.3] pg/ml, p=0.03). Among other observations, there was an increase in the concentration of IL-1β after the exposition with HC-030031, as well as a decrease in TNF-α, IFN-γ and IL-12p70 after the treatment with CA. CSE caused a minor inhibition in phagocytic cells number, which was not prevented by TRPA1 blocking. CA, on the contrary, increased the phagocytic activity of macrophages. The initial expression of TRPA1 had a negative correlation with the dynamics of CXCL10 in response to CSE and CA but a positive correlation with the number of phagocytic cells after exposition with CA (ρ=0.81, p=0.005). Conclusions. TRPA1 expressed on macrophages apparently mediate an anti-inflammatory effect in terms of produced cytokines but increase phagocytic activity of the cells. TRPA1 are also major receptors involved in the diminished CXCL10 production by macrophage under exposition with cigarette smoke

Combining Topical Agonists With the Recording of Event-Related Brain Potentials to Probe the Functional Involvement of TRPM8, TRPA1 and TRPV1 in Heat and Cold Transduction in the Human Skin

TRP channels play a central role in the transduction of thermal and nociceptive stimuli by free nerve endings. Most of the research on these channels has been conducted in vitro or in vivo in nonhuman animals and translation of these results to humans must account for potential experimental biases and interspecific differences. This study aimed at evaluating the involvement of TRPM8, TRPA1 and TRPV1 channels in the transduction of heat and cold stimuli by the human thermonociceptive system. For this purpose, we evaluated the effects of topical agonists of these 3 channels (menthol, cinnamaldehyde and capsaicin) on the event-related brain potentials (ERPs) elicited by phasic thermal stimuli (target temperatures: 10°C, 42°C, and 60°C) selected to activate cold Aδ thermoreceptors, warm sensitive C thermoreceptors and heat sensitive Aδ polymodal nociceptors. Sixty-four participants were recruited, 16 allocated to each agonist solution group (20% menthol, 10% cinnamaldehyde, .025% capsaicin and 1% capsaicin). Participants were treated sequentially with the active solution on one forearm and vehicle only on the other forearm for 20 minutes. Menthol decreased the amplitude and increased the latency of cold and heat ERPs. Cinnamic aldehyde decreased the amplitude and increased the latency of heat but not cold ERPs. Capsaicin decreased the amplitude and increased the latency of heat ERPs and decreased the amplitude of the N2P2 complex of the cold ERPs without affecting the earlier N1 wave or the latencies of the peaks. These findings are compatible with previous evidence indicating that TRPM8 is involved in innocuous cold transduction and that TRPV1 and TRPA1 are involved in noxious heat transduction in humans. PerspectiveBy chemically modulating TRPM8, TRPA1 and TRPV1 reactivity (key molecules in the transduction of temperature) and assessing how this affected EEG responses to the activation of cold thermoreceptors and heat nociceptors, we aimed at confirming the role of these channels in a functional healthy human model.

Proton Sensing on the Ocular Surface: Implications in Eye Pain.

Protons reaching the eyeball from exogenous acidic substances or released from damaged cells during inflammation, immune cells, after tissue injury or during chronic ophthalmic conditions, activate or modulate ion channels present in sensory nerve fibers that innervate the ocular anterior surface. Their identification as well as their role during disease is critical for the understanding of sensory ocular pathophysiology. They are likely to mediate some of the discomfort sensations accompanying several ophthalmic formulations and may represent novel targets for the development of new therapeutics for ocular pathologies. Among the ion channels expressed in trigeminal nociceptors innervating the anterior surface of the eye (cornea and conjunctiva) and annex ocular structures (eyelids), members of the TRP and ASIC families play a critical role in ocular acidic pain. Low pH (pH 6) activates TRPV1, a polymodal ion channel also activated by heat, capsaicin and hyperosmolar conditions. ASIC1, ASIC3 and heteromeric ASIC1/ASIC3 channels present in ocular nerve terminals are activated at pH 7.2–6.5, inducing pain by moderate acidifications of the ocular surface. These channels, together with TRPA1, are involved in acute ocular pain, as well as in painful sensations during allergic keratoconjunctivitis or other ophthalmic conditions, as blocking or reducing channel expression ameliorates ocular pain. TRPV1, TRPA1 and other ion channels are also present in corneal and conjunctival cells, promoting inflammation of the ocular surface after injury. In addition to the above-mentioned ion channels, members of the K2P and P2X ion channel families are also expressed in trigeminal neurons, however, their role in ocular pain remains unclear to date. In this report, these and other ion channels and receptors involved in acid sensing during ocular pathologies and pain are reviewed.

Prostaglandin E2 sensitizes the cough reflex centrally via EP3 receptor-dependent activation of NaV 1.8 channels

BackgroundCough hypersensitivity is a major characteristic feature associated with several types of cough, including chronic cough, but its underlying mechanisms remain to be fully understood. Inflammatory mediators, such as prostaglandin E2 (PGE2), have been implicated in both peripheral induction and sensitization of the cough reflex. In this study, using a conscious guinea pig model of cough, we investigated whether PGE2 can sensitize the cough reflex via central actions and, if so, via which mechanisms.MethodsAll drugs were administered by intracerebroventricular (i.c.v.) route and whole-body plethysmograph set-up was used for both induction, using aerosolized citric acid (0.2 M), and recording of cough. Immunohistochemistry was performed to confirm the expression of NaV 1.8 channels in the nucleus tractus solitarius (nTS).ResultsWe show that both PGE2 and the non-selective EP1/EP3 agonist, sulprostone, dose-dependently enhanced the citric acid-induced cough (P ≤ 0.001, P ≤ 0.01, respectively). Pretreatment with the EP1 antagonist, ONO-8130, did not affect the sulprostone-induced cough sensitization, whilst the EP3 antagonist, L-798,106, dose-dependently inhibited this effect (P ≤ 0.05). Furthermore, treatment with either the EP2 agonist, butaprost or the EP4 agonist, L-902,688, had no effect on cough sensitization. Additionally, pretreatment with either the TRPV1 antagonist, JNJ-17203212 or the TRPA1 antagonist, HC-030031, alone or in combination, nor with the NaV 1.1, 1.2, 1.3, 1.4, 1.6 and 1.7 channel blocker, tetrodotoxin, had any effect on the cough. In contrast, pretreatment with the NaV 1.8 antagonist, A-803467, dose-dependently inhibited this effect (P ≤ 0.05). Furthermore, NaV 1.8 channels were shown to be expressed in the nTS.ConclusionCollectively, our findings show that PGE2 sensitizes the cough reflex centrally via EP3 receptor-dependent activation of NaV 1.8 but independently of TRPV1,TRPA1 and TTX-sensitive sodium channel activation. These results indicate that PGE2 plays an important role in central sensitization of the cough reflex and suggest that central EP3 receptors and/or NaVv 1.8 channels may represent novel antitussive molecular targets.Graphical

Synthesis, structural and spectroscopic characterization, in silico study, and antinociceptive effect in adult zebrafish of 2-(4-isobutylphenyl) -N'-phenylpropanohydrazide

• Structural characterization of a novel ibuprofen derivative. • Vibrational analysis was carried out. • Electronic absorption spectrum was obtained. • Molecular docking and ADMET studies were carried out. • The novel compound was not toxic, and affected locomotor activity of the Zebrafish. In this work, an ibuprofen derivative 2-(4-isobutylphenyl) -N'-phenylpropanohydrazide (ACPHZN) was synthesized and characterized by NMR, ATR-FTIR, FT-Raman, and UV-Vis spectroscopy, while their structural and spectroscopic properties were investigated using DFT calculations. In vivo study using animal model in an adult Zebrafish ( Danio rerio ), and molecular docking was performed. In addition, molecular descriptors of the properties of absorption, distribution, metabolism and excretion, and toxicity (ADMET) were obtained. The data calculated for ibuprofen derivative are in accordance with the experimental values. From the assays in adult zebrafish, it was found that the ibuprofen derivative was non-toxic and exhibited analgesic properties through the TRPA1, TRPV1 and TRPM8 channels. Molecular docking revealed six interactions of ACPHZN with residues of the capsaicin binding site, and a more favorable affinity energy (-9.0 kcal / mol). ADMET studies suggest that ACPHZN has a pharmacological principle as an oral drug based on a longer half-life in the human body.

Trpv1 and Trpa1 are not essential for Psickle-like activity in red cells of the SAD mouse model of sickle cell disease

The molecular identity of Psickle, the deoxygenation-activated cation conductance of the human sickle erythrocyte, remains unknown. We observed in human sickle red cells that inhibitors of TRPA1 and TRPV1 inhibited Psickle, whereas a TRPV1 agonist activated a Psickle-like cation current. These observations prompted us to test the roles of TRPV1 and TRPA1 in Psickle in red cells of the SAD mouse model of sickle cell disease. We generated SAD mice genetically deficient in either TRPV1 or TRPA1. SAD;Trpv1−/− and SAD;Trpa1−/− mice were indistinguishable in appearance, hematological indices, and osmotic fragility from SAD mice. We found that deoxygenation-activated cation currents remained robust in SAD;Trpa1−/− and SAD;Trpv1−/− mice. In addition, 45Ca2+ influx into SAD mouse red cells during prolonged deoxygenation was not reduced in red cells from SAD;Trpa1−/− and SAD;Trpv1−/− mice. We conclude that the nonspecific cation channels TRPA1 and TRPV1 are not required for deoxygenation to stimulate Psickle-like activity in red cells of the SAD mouse model of sickle cell disease.(159)

TRPA1 Expression and Pathophysiology in Immune Cells.

The non-selective cation channel TRPA1 is best known as a broadly-tuned sensor expressed in nociceptive neurons, where it plays key functions in chemo-, thermo-, and mechano-sensing. However, in this review we illustrate how this channel is expressed also in cells of the immune system. TRPA1 has been detected, mainly with biochemical techniques, in eosinophils, mast cells, macrophages, dendritic cells, T cells, and B cells, but not in neutrophils. Functional measurements, in contrast, remain very scarce. No studies have been reported in basophils and NK cells. TRPA1 in immune cells has been linked to arthritis (neutrophils), anaphylaxis and atopic dermatitis (mast cells), atherosclerosis, renal injury, cardiac hypertrophy and inflammatory bowel disease (macrophages), and colitis (T cells). The contribution of TRPA1 to immunity is dual: as detector of cell stress, tissue injury, and exogenous noxious stimuli it leads to defensive responses, but in conditions of aberrant regulation it contributes to the exacerbation of inflammatory conditions. Future studies should aim at characterizing the functional properties of TRPA1 in immune cells, an essential step in understanding its roles in inflammation and its potential as therapeutic target.

Activation of Astrocytes in the Persistence of Post-hypoxic Respiratory Augmentation.

Acute hypoxia increases ventilation. After cessation of hypoxia loading, ventilation decreases but remains above the pre-exposure baseline level for a time. However, the mechanism of this post-hypoxic persistent respiratory augmentation (PHRA), which is a short-term potentiation of breathing, has not been elucidated. We aimed to test the hypothesis that astrocytes are involved in PHRA. To this end, we investigated hypoxic ventilatory responses by whole-body plethysmography in unanesthetized adult mice. The animals breathed room air, hypoxic gas mixture (7% O2, 93% N2) for 2min, and again room air for 10min before and after i.p. administration of low (100mg/kg) and high (300mg/kg) doses of arundic acid (AA), an astrocyte inhibitor. AA suppressed PHRA, with the high dose decreasing ventilation below the pre-hypoxic level. Further, we investigated the role of the astrocytic TRPA1 channel, a putative ventilatory hypoxia sensor, in PHRA using astrocyte-specific Trpa1 knockout (asTrpa1−/−) and floxed Trpa1 (Trpa1f/f) mice. In both Trpa1f/f and asTrpa1−/− mice, PHRA was noticeable, indicating that the astrocyte TRPA1 channel was not directly involved in PHRA. Taken together, these results indicate that astrocytes mediate the PHRA by mechanisms other than TRPA1 channels that are engaged in hypoxia sensing.

Heat shock factor 1 in brain tumors: a link with transient receptor potential channels TRPV1 and TRPA1.

Novel data report a "cross-talk" between Heat-Shock Factor 1 (HSF1) and the transient receptor potential vanilloid 1 cation channel (TRPV1) located in the cell membrane, introducing these channels as possible drug targets for the regulation of HSF1 activation. This study aims to investigate the co-expression of TRPV1 and HSF1 in human brain tumors. Additionally, the expression of the transient receptor potential ankyrin 1 channel (TRPA1), which is co-operated with TRPV1 in a plethora of cells, was studied. Immunohistochemical staining for HSF1, TRPV1 and TRPA1 expression was quantitatively analyzed in paraffin-embedded semi-serial tissue sections from 74 gliomas and 71 meningiomas. mRNA levels of HSF1, TRPV1 and TRPA1 were evaluated using real-time PCR. Although HSF1 was significantly increased compared with TRPV1/TRPA1 (p ≤ 0.001) in both gliomas and meningiomas, high co-expression levels for HSF1, TRPV1 and TRPA1 were found in 62.50% of diffuse fibrillary astrocytomas (WHO, grade II), 37.50% of anaplastic astrocytomas (WHO, grade III), 16.32% of glioblastomas multiforme (WHO, grade IV), and 42.25% of meningiomas (WHO, grade I and II). Correlation analysis revealed a relationship of HSF1 with TRPV1/TRPA1 in diffuse fibrillary astrocytomas (WHO, grade II) and benign meningiomas (WHO, grade I) contrary to glioblastomas multiforme (WHO, grade IV) and high grade meningiomas (WHO, grade II). Importantly, TRPA1 and TRPV1 expression levels were significantly increased in meningiomas compared with astrocytic tumors (p < 0.05). In conclusion, HSF1 and TRPV1/TRPA1 co-expression may be implicated in the pathogenesis of human brain tumors and should be considered for the therapeutic approaches for these tumors.

Metformin strengthens uroepithelial immunity against E. coli infection

Urinary tract infection frequently caused by E. coli is one of the most common bacterial infections. Increasing antibiotic resistance jeopardizes successful treatment and alternative treatment strategies are therefore mandatory. Metformin, an oral antidiabetic drug, has been shown to activate macrophages in the protection against certain infecting microorganisms. Since epithelial cells often form the first line of defense, we here investigated the effect on uroepithelial cells during E. coli infection. Metformin upregulated the human antimicrobial peptides cathelicidin LL-37 and RNase7 via modulation of the TRPA1 channel and AMPK pathway. Interestingly, metformin stimulation enriched both LL-37 and TRPA1 in lysosomes. In addition, metformin specifically increased nitric oxide and mitochondrial, but not cytosolic ROS. Moreover, metformin also triggered mRNA expression of the proinflammatory cytokines IL1B, CXCL8 and growth factor GDF15 in human uroepithelial cells. The GDF15 peptide stimulated macrophages increased LL-37 expression, with increased bacterial killing. In conclusion, metformin stimulation strengthened the innate immunity of uroepithelial cells inducing enhanced extracellular and intracellular bacterial killing suggesting a favorable role of metformin in the host defense.

Nerve-associated transient receptor potential ion channels can contribute to intrinsic resistance to bacterial adhesion in vivo.

The cornea of the eye differs from other mucosal surfaces in that it lacks a viable bacterial microbiome and by its unusually high density of sensory nerve endings. Here, we explored the role of corneal nerves in preventing bacterial adhesion. Pharmacological and genetic methods were used to inhibit the function of corneal sensory nerves or their associated transient receptor potential cation channels TRPA1 and TRPV1. Impacts on bacterial adhesion, resident immune cells, and epithelial integrity were examined using fluorescent labeling and quantitative confocal imaging. TRPA1/TRPV1 double gene-knockout mice were more susceptible to adhesion of environmental bacteria and to that of deliberately-inoculated Pseudomonas aeruginosa. Supporting the involvement of TRPA1/TRPV1-expressing corneal nerves, P. aeruginosa adhesion was also promoted by treatment with bupivacaine, or ablation of TRPA1/TRPV1-expressing nerves using RTX. Moreover, TRPA1/TRPV1-dependent defense was abolished by enucleation which severs corneal nerves. High-resolution imaging showed normal corneal ultrastructure and surface-labeling by wheat-germ agglutinin for TRPA1/TRPV1 knockout murine corneas, and intact barrier function by absence of fluorescein staining. P. aeruginosa adhering to corneas after perturbation of nerve or TRPA1/TRPV1 function failed to penetrate the surface. Single gene-knockout mice showed roles for both TRPA1 and TRPV1, with TRPA1-/- more susceptible to P. aeruginosa adhesion while TRPV1-/- corneas instead accumulated environmental bacteria. Corneal CD45+/CD11c+ cell responses to P. aeruginosa challenge, previously shown to counter bacterial adhesion, also depended on TRPA1/TRPV1 and sensory nerves. Together, these results demonstrate roles for corneal nerves and TRPA1/TRPV1 in corneal resistance to bacterial adhesion in vivo and suggest that the mechanisms involve resident immune cell populations.

Transient Receptor Potential channels, TRPV1 and TRPA1 in melanocytes synergize UV-dependent and UV-independent melanogenesis.

Melanogenesis is essential for pigmentation and deregulated melanogenesis causes pigmentary diseases. Psoralen and ultraviolet A (PUVA) therapy strongly stimulates pigmentation, but the underlying molecular mechanisms are elusive. Melanin content of cultured human melanocytes was spectrophotometrically measured. Patch-clamp recordings were made in human melanocytes or HEK 293 cells transiently expressing wild type or mutant human TRPV1 and TRPA1 channels. Endogenous expression of TRPV1 and TRPA1 in melanocytes was analysed by western blotting and was knocked down with siRNA. In vivo pigmentary responses were measured by a colorimeter in mouse ear skin. The expression of TRPV1 and TRPA1 in human pigmented lesions was examined by immunohistochemical staining. PUVA strongly stimulated melanogenesis and PUVA-induced TRPV1 and TRPA1 channel activation in melanocytes and the resulting Ca2+ influx were required for the stimulated melanogenesis both in vitro and in vivo. Agonists-induced TRPV1 and TRPA1 activation alone did not stimulate melanogenesis, but it synergized UVA or intrinsic cAMP and NO signalling pathways to stimulate UV-dependent or UV-independent melanogenesis. Moreover, the expressions of TRPV1 and TRPA1 were increased in human melanocytic lesions and inhibition of both channels decreased melanin content in melanoma cells. TRPV1 and TRPA1 are key molecular sensors and enhancers of extrinsic and intrinsic melanogenic signals in both physiological and pathological conditions, and activation of both channels in melanocytes contributes to PUVA therapy-induced pigmentation. Our work provides a common mechanism of melanogenic regulation and highlights TRPV1 and TRPA1 as potential therapeutic targets for pigmentary disorders.

Functional expression of TRPA1 channel, TRPV1 channel and TMEM100 in human odontoblasts.

TRPA1 and TRPV1 channels respond to external stimulation as pain mediators and form a complex with a transmembrane protein TMEM100 in some tissues. However, their expression and interaction in dental pulp is unclear. To investigate the functional co-expression of TRPA1 channel, TRPV1 channel and TMEM100 in human odontoblasts (HODs), immunohistochemistry, immunofluorescence staining and Western blot were used to study their co-localization and expression in both native HODs and cultured HOD-like cells. Calcium imaging was used to detect the functional interaction between TRPA1 and TRPV1 channels. Immunohistochemistry and multiple immunofluorescence staining of tooth slices showed positive expression of TRPA1 channel, TRPV1 channel and TMEM100 mainly in the cell bodies of HODs, and TRPA1 channel presented more obvious immunofluorescence in the cell processes than TRPV1 channel and TMEM100. HALO software analysis showed that TRPA1 and TRPV1 channels were positively expressed in most TMEM100+ HODs and these three proteins were strongly correlated in HODs (P<0.01). The protein expression levels of TRPA1 channel, TRPV1 channel and TMEM100 in HODs showed no significant difference (P > 0.05). Double immunofluorescence staining of cultured HOD-like cells visually demonstrated that TRPA1 and TRPV1 channel were both highly co-localized with TMEM100 with similar expressive intensity. Calcium imaging showed that there was a functional interaction between TRPA1 and TRPV1 channels in HOD-like cells, and TRPA1 channel might play a greater role in this interaction. Overall, we concluded that TRPA1 channel, TRPV1 channel and TMEM100 could be functionally co-expressed in HODs.

In silico Identification of Key Factors Driving the Response of Muscle Sensory Neurons to Noxious Stimuli.

Nociceptive nerve endings embedded in muscle tissue transduce peripheral noxious stimuli into an electrical signal [i.e., an action potential (AP)] to initiate pain sensations. A major contributor to nociception from the muscles is mechanosensation. However, due to the heterogeneity in the expression of proteins, such as ion channels, pumps, and exchangers, on muscle nociceptors, we currently do not know the relative contributions of different proteins and signaling molecules to the neuronal response due to mechanical stimuli. In this study, we employed an integrated approach combining a customized experimental study in mice with a computational model to identify key proteins that regulate mechanical nociception in muscles. First, using newly collected data from somatosensory recordings in mouse hindpaw muscles, we developed and then validated a computational model of a mechanosensitive mouse muscle nociceptor. Next, by performing global sensitivity analyses that simulated thousands of nociceptors, we identified three ion channels (among the 17 modeled transmembrane proteins and four endoplasmic reticulum proteins) as potential regulators of the nociceptor response to mechanical forces in both the innocuous and noxious range. Moreover, we found that simulating single knockouts of any of the three ion channels, delayed rectifier voltage-gated K+ channel (Kv1.1) or mechanosensitive channels Piezo2 or TRPA1, considerably altered the excitability of the nociceptor (i.e., each knockout increased or decreased the number of triggered APs compared to when all channels were present). These results suggest that altering expression of the gene encoding Kv1.1, Piezo2, or TRPA1 might regulate the response of mechanosensitive muscle nociceptors.