Expression Of Transient Receptor Potential A1 Research Articles

An experimental study of acute toxicity and pharmacology of fermented Platycodonis Radix

This study investigated the acute toxicity of fermented Platycodonis Radix on mice and its effect on coughing in mice infected with Mycoplasma pneumoniae. The maximum dosage(MAD) was used in the acute toxicity experiment on mice to observe the signs of mice. After 14 days, dissection, blood biochemical examination, and pathological tissue section observation were conducted. In the pharmacological experiment of fermented Platycodonis Radix, 60 healthy BALB/c mice, 30 males and 30 females, were randomly divided into a blank group, a model group, a carbetapentane group(0.013 g·kg~(-1)·d~(-1)), and high-, medium-, and low-dose fermented Platycodonis Radix groups(5.2, 2.6, and 1.3 g·kg~(-1)·d~(-1)), with 10 mice in each group. Except for the blank group, the mice in the other five groups underwent model induction by intranasally instilling 20 μL of 1×10~6 CCU M. pneumoniae for 3 days, and the mice in each group were orally administered the corresponding drugs for 7 days. Cough induction experiment was conducted to observe and record the cough latency and total cough count within 3 min for each group. Hematoxylin-eosin(HE) staining and Masson staining were used to observe the pathological changes in lung tissues. Immunohistochemistry was performed to observe the protein expression of transient receptor potential A1(TRPA1), calcitonin gene-related peptide(CGRP), and substance P(SP) in the lung tissues of mice in each group. Real-time fluorescence-based quantitative polymerase chain reaction(qRT-PCR) was used to elucidate the changes in the mRNA levels of cough-related factors TRPA1, CGRP, and SP in mice treated with fermented Platycodonis Radix. No mice died in the acute toxicity experiment, and there were no changes in general behavior and major organ histopathological examinations. Compared with the blank group, there were no statistically significant differences in blood biochemical indexes. In the pharmacological experiment of fermented Platycodonis Radix, compared with the model group, the high-and medium-dose fermented Platycodonis Radix groups showed improved lung tissue structure of mice, with clear structure and regular tissue morphology. The qRT-PCR and immunohistochemical detection showed a decrease in the expression of TRPA1, CGRP, and SP in the fermented Platycodonis Radix groups. Fermented Platycodonis Radix can exert an inhibitory effect on cough by suppressing the expression of TRPA1, CGRP, and SP in lung tissues, thereby identifying the target of the drug.

Repeated exposure to temperature variation exacerbates airway inflammation through TRPA1 in a mouse model of asthma.

Studies from epidemiology suggest that ambient temperature is one of the underlying triggers and potential causes of asthma. The aim of this study was to examine the impact and the molecular mechanism of temperature-invoked airway inflammation using an experimental model of asthma in BALB/c mice. Mice were exposed to different temperature conditions (steady 26°C, 26°C/18°C cycle, 26°C/10°C cycle) and received sensitization and challenge of ovalbumin (OVA) during a 21-day period. HC030031, a selective transient receptor potential A1 (TRPA1) channel blocker, was used to investigate the underlying mechanism of TRPA1 in 'asthmatic' airways. After the final OVA challenge, in vivo lung function was measured, and bronchoalveolar lavage fluid (BALF) and pulmonary inflammation were assessed. The temperature variations, especially the largest temperature difference (16°C), exacerbated airway inflammation in OVA-induced mice, increasing the levels of serum total-IgE (immunoglobulin E) and IgG1, inflammatory cells and cytokines in BALF. Analysis of histopathological changes and lung function verified that repeated exposure to very cold and changed temperatures aggravated airway hyperresponsiveness (AHR). Significant upregulation of TRPA1 expression was revealed by immunohistochemistry in the presence of the largest temperature variation (26°C/10°C cycle), while administration of HC030031 successfully inhibited TRPA1 expression, thus attenuating the asthma-like pathological features. Repeated exposure to temperature variation exacerbated experimental 'asthma' and TRPA1 mediated this temperature-dependent inflammatory effect.

A curcumin derivative J147 ameliorates diabetic peripheral neuropathy in streptozotocin (STZ)-induced DPN rat models through negative regulation AMPK on TRPA1

To investigate the specific molecular mechanisms and effects of curcumin derivative J147 on diabetic peripheral neuropathy (DPN). We constructed streptozotocin (STZ)-induced DPN rat models to detected mechanical withdrawal threshold (MWT) in vivo using Von Frey filaments. In vitro, we measured cell viability and apoptosis, adenosine 5'-monophosphate-activated protein kinase (AMPK) and transient receptor potential A1 (TRPA1) expression using MTT, flow cytometry, qRT-PCR and western blot. Then, TRPA1 expression level and calcium reaction level were assessed in agonist AICAR treated RSC96cells. The results showed that J147reduced MWT in vivo, increased the mRNA and protein level of AMPK, reduced TRPA1 expression and calcium reaction level in AITCR treated RSC96 cells, and had no obvious effect on cell viability and apoptosis. Besides, AMPK negative regulated TRPA1 expression in RSC96 cells. J147 could ameliorate DPN via negative regulation AMPK on TRPA1 in vivo and in vitro. A curcumin derivative J147might be a new therapeutic potential for the treatment of DPN.

TRPA1 channels: expression in non-neuronal murine lung tissues and dispensability for hyperoxia-induced alveolar epithelial hyperplasia.

Transient receptor potential A1 (TRPA1) channels were originally characterized in neuronal tissues but also identified in lung epithelium by staining with fluorescently coupled TRPA1 antibodies. Its exact function in non-neuronal tissues, however, is elusive. TRPA1 is activated in vitro by hypoxia and hyperoxia and is therefore a promising TRP candidate for sensing hyperoxia in pulmonary epithelial cells and for inducing alveolar epithelial hyperplasia. Here, we isolated tracheal, bronchial, and alveolar epithelial cells and show low but detectable TRPA1 mRNA levels in all these cells as well as TRPA1 protein by Western blotting in alveolar type II (AT II) cells. We quantified changes in intracellular Ca2+ ([Ca2+]i) levels induced by application of hyperoxic solutions in primary tracheal epithelial, bronchial epithelial, and AT II cells isolated from wild-type (WT) and TRPA1-deficient (TRPA1-/-) mouse lungs. In all cell types, we detected hyperoxia-induced rises in [Ca2+]i levels, which were not significantly different in TRPA1-deficient cells compared to WT cells. We also tested TRPA1 function in a mouse model for hyperoxia-induced alveolar epithelial hyperplasia. A characteristic significant increase in thickening of alveolar tissues was detected in mouse lungs after exposure to hyperoxia, but not in normoxic WT and TRPA1-/- controls. Quantification of changes in lung morphology in hyperoxic WT and TRPA1-/- mice, however, again revealed no significant changes. Therefore, TRPA1 expression does neither appear to be a key player for hyperoxia-induced changes in [Ca2+]i levels in primary lung epithelial cells, nor being essential for the development of hyperoxia-induced alveolar epithelial hyperplasia.

Mechanisms of the adenosine A2A receptor-induced sensitization of esophageal C fibers.

Clinical studies indicate that adenosine contributes to esophageal mechanical hypersensitivity in some patients with pain originating in the esophagus. We have previously reported that the esophageal vagal nodose C fibers express the adenosine A2A receptor. Here we addressed the hypothesis that stimulation of the adenosine A2A receptor induces mechanical sensitization of esophageal C fibers by a mechanism involving transient receptor potential A1 (TRPA1). Extracellular single fiber recordings of activity originating in C-fiber terminals were made in the ex vivo vagally innervated guinea pig esophagus. The adenosine A2A receptor-selective agonist CGS21680 induced robust, reversible sensitization of the response to esophageal distention (10-60 mmHg) in a concentration-dependent fashion (1-100 nM). At the half-maximally effective concentration (EC50: ≈3 nM), CGS21680 induced an approximately twofold increase in the mechanical response without causing an overt activation. This sensitization was abolished by the selective A2A antagonist SCH58261. The adenylyl cyclase activator forskolin mimicked while the nonselective protein kinase inhibitor H89 inhibited mechanical sensitization by CGS21680. CGS21680 did not enhance the response to the purinergic P2X receptor agonist α,β-methylene-ATP, indicating that CGS21680 does not nonspecifically sensitize to all stimuli. Mechanical sensitization by CGS21680 was abolished by pretreatment with two structurally different TRPA1 antagonists AP18 and HC030031. Single cell RT-PCR and whole cell patch-clamp studies in isolated esophagus-specific nodose neurons revealed the expression of TRPA1 in A2A-positive C-fiber neurons and demonstrated that CGS21682 potentiated TRPA1 currents evoked by allylisothiocyanate. We conclude that stimulation of the adenosine A2A receptor induces mechanical sensitization of nodose C fibers by a mechanism sensitive to TRPA1 antagonists indicating the involvement of TRPA1.

Drosophila TRPA1 isoforms detect UV light via photochemical production of H2O2

The transient receptor potential A1 (TRPA1) channel is an evolutionarily conserved detector of temperature and irritant chemicals. Here, we show that two specific isoforms of TRPA1 in Drosophila are H2O2 sensitive and that they can detect strong UV light via sensing light-induced production of H2O2. We found that ectopic expression of these H2O2-sensitive Drosophila TRPA1 (dTRPA1) isoforms conferred UV sensitivity to light-insensitive HEK293 cells and Drosophila neurons, whereas expressing the H2O2-insensitive isoform did not. Curiously, when expressed in one specific group of motor neurons in adult flies, the H2O2-sensitive dTRPA1 isoforms were as competent as the blue light-gated channelrhodopsin-2 in triggering motor output in response to light. We found that the corpus cardiacum (CC) cells, a group of neuroendocrine cells that produce the adipokinetic hormone (AKH) in the larval ring gland endogenously express these H2O2-sensitive dTRPA1 isoforms and that they are UV sensitive. Sensitivity of CC cells required dTRPA1 and H2O2 production but not conventional phototransduction molecules. Our results suggest that specific isoforms of dTRPA1 can sense UV light via photochemical production of H2O2. We speculate that UV sensitivity conferred by these isoforms in CC cells may allow young larvae to activate stress response--a function of CC cells--when they encounter strong UV, an aversive stimulus for young larvae.

The role of transient receptor potential A 1 (TRPA1) in the development and maintenance of carrageenan-induced hyperalgesia

Transient receptor potential ankyrin 1 (TRPA1) is a nonselective cation channel important in setting nociceptive threshold. It is expressed in nociceptive C-fibers and in non-neuronal cells involved in pro-inflammatory mediators' release. We asked whether TRPA1 contributes to carrageenan-induced hyperalgesia in rats, and if so, whether this contribution is mediated by mechanisms involved in inflammation such as cytokine release and neutrophil migration and/or by a direct sensitization of the primary afferent nociceptors. Pharmacological blockade of local TRPA1 by its selective antagonist HC 030031 prevented and reversed carrageenan-induced hyperalgesia, which was detected either by a mechanical or chemical (low dose of capsaicin) stimulus. However, it did not affect either carrageenan-induced cytokines expression or neutrophil migration. The neuronal TRPA1 gene silencing induced by intrathecal pre-treatment with antisense oligodoexynucleotide completely prevented carrageenan-induced hyperalgesia over 24 h and significantly reduced TRPA1 expression in the dorsal root ganglia cells (L5-6), which was not affected by carrageenan treatment. We conclude that TRPA1 plays an important role in the development and maintenance of carrageenan-induced inflammatory hyperalgesia by directly contributing to nociceptor excitability.

Abstract 385: TRPA1 Mediates Angiotensinogen Augmentation in Renal Proximal Tubular Cells

Overactivity of the intrarenal renin-angiotensin system contributes to the development of hypertension and renal injury. In this process, regulation of angiotensinogen (AGT) in renal proximal tubular cells (RPTC) is a key factor. Oxidative stress stimulates AGT expression accompanied by activation of p38 MAPK in RPTC. Transient receptor potential A1 (TRPA1) is known to be a sensor activated by a variety of noxious stimuli including hydrogen peroxide. Activated TRPA1 induces calcium influx across the plasma membrane leading to activation of AGT-inducible signal transducers. However, the presence and function of TRPA1 in the kidney have not been delineated. Therefore, this study was performed to demonstrate expression of intrarenal TRPA1 and its role in AGT augmentation in RPTC. Expression of TRPA1 in mouse kidney and cultured mouse RPTC were determined by RT-PCR, in situ hybridization, western blot analysis and immunocytochemistry. The RPTC were treated with 100 μM H2O2 for 1 hr with or without 10μM HC030031, a TRPA1 specific antagonist. EGTA was used to the culture medium for depletion of extracellular calcium. Thereafter, AGT expression levels were evaluated by real-time RT-PCR, and TRPA1 expression levels and p38 MAPK activity were determined by western blot analyses. TRPA1 expression was observed in tubules of renal cortex, and the cultured PRTC expressed TRPA1 mRNA and protein. H2O2 increased AGT expression (1.54 ± 0.08, ratio to control) in RPTC. The AGT augmentation was suppressed by HC030031 (1.19 ± 0.05, ratio to control). Calcium depletion also resulted in attenuation of the AGT augmentation induced by H2O2. Although H2O2 induced phosphorylation of p38 MAPK, HC030031 did not inhibit the p38 MAPK activation. TRPA1 expression was increased by H2O2 under the experimental condition (1.90 ± 0.06, ratio to control). These results suggest that TRPA1 is expressed in RPTC, which contributes to H2O2-induced AGT augmentation via calcium-dependent but p38 MAPK-independent pathways. Furthermore, increase in TRPA1 expression by H2O2 likely facilitates the stimulation of AGT expression. Therefore, TRPA1 may play an important role in the progression of oxidative stress-associated renal injury and hypertension via the stimulation of AGT expression in RPTC.

TRPA1‐activated anion secretion is linked with PGE2 receptor EP4 in human and rat colon

The transient receptor potential A1 (TRPA1) channel is considered a chemosensor in several sensory tissues. In the present study, we investigated the secretory effect of potential TRPA1 agonist allyl isothiocyanate (AITC) in rat and human colons using the Ussing flux chamber. The mucosal application of AITC (10−6–10−3 M) induced Cl− and HCO3 − secretion in a concentration‐dependent manner, while the serosal application induced weaker effect. AITC‐evoked anion secretion was attenuated by the pretreatment with cyclooxygenase inhibitor piroxicam and prostaglandin (PG) E2, but the secretion was not affected by tetrodotoxin, atropine, or extracellular Ca2+ depletion. These results indicated that TRPA1 activation induces anion secretion through PG synthesis independent of neural pathway in the colon. Further analysis also indicated that AITC‐evoked anion secretion is mainly mediated by EP4. In addition, RT‐PCR using isolated colonic crypts, in situ hybridization and immunohistochemistry supported the TRPA1 expression in epithelial cells. Therefore, we conclude that the activation of TRPA1 activity on the colonic epithelial cells is likely involved in host defense mechanism through rapid anion secretion.

Activation of TRPA1 by luminal stimuli induces EP4-mediated anion secretion in human and rat colon

In gastrointestinal (GI) physiology, anion and fluid secretion is an important function for host defense and is induced by changes in the luminal environment. The transient receptor potential A1 (TRPA1) channel is considered to be a chemosensor in several sensory tissues. Although the function of TRPA1 has been studied in GI motility, its contribution to the transepithelial ion transport system has rarely been discussed. In the present study, we investigated the secretory effect of the potential TRPA1 agonist allyl isothiocyanate (AITC) in rat and human colon using an Ussing chamber. The mucosal application of AITC (10(-6)-10(-3) M) induced Cl(-) and HCO(3)(-) secretion in a concentration-dependent manner, whereas the serosal application induced a significantly weaker effect. AITC-evoked anion secretion was attenuated by tissue pretreatment with piroxicam and prostaglandin (PG) E(2); however, this secretion was not affected by TTX, atropine, or extracellular Ca(2+) depletion. These experiments indicate that TRPA1 activation induces anion secretion through PG synthesis, independent of neural pathways in the colon. Further analysis also indicates that AITC-evoked anion secretion is mediated mainly by the EP(4) receptor subtype. The magnitude of the secretory response exhibited segmental heterogeneity in rat colon. Real-time PCR analysis showed the segmental difference was corresponding to the differential expression of EP(4) receptor and cyclooxygenase-1 and -2. In addition, RT-PCR, in situ hybridization, and immunohistochemical studies showed TRPA1 expression in the colonic epithelia. Therefore, we conclude that the activation of TRPA1 in colonic epithelial cells is likely involved in the host defense mechanism through rapid anion secretion.

Overexpression of Human Transient Receptor Potential M5 Upregulates Endogenous Human Transient Receptor Potential A1 in a Stable HEK Cell Line

Transient receptor potential M5 (TRPM5), a monovalent cation channel, is primarily activated by increases in intracellular calcium. However, we found unexpectedly that allyl isothiocyanate (AITC) and structural analogs triggered a membrane potential and calcium dye responses in TRPM5-HEK cells (AITC EC₅₀ = 9.0 ± 2.4 μM, n = 5). Although AITC and its analogs were more potent on transient receptor potential A1 (TRPA1)-HEK cells (AITC EC₅₀ = 0.23 ± 0.03 μM, n = 4), the rank order potency of these compounds were similar for TRPM5- and TRPA1-HEK cells. No response to these compounds was seen in parental HEK cells, TRPM5-CHO cells, and TRPM4b-, TRPM8-, or TRPV1-transfected HEK cells. An AITC-evoked current in TRPM5-HEK cells was confirmed in whole-cell voltage clamp recording. AITC elicited an intracellular calcium increase that was not dependent on phorpholipase C(β)₂ (PLC(β)₂) activation but was dependent on extracellular calcium concentration. TRPA1 mRNA was upregulated fourfold in TRPM5-HEK cells compared with parental cells. In contrast, TRPA1 was not upregulated in HEK cells transfected in a similar manner with TRPV1 or TRPM8 genes. The AITC response was blocked by a TRPA1 inhibitor and reduced by a TRPM5 inhibitor and by targeted TRPA1 siRNA. These results suggest that TRPM5 may play a role in upregulating endogenous expression of TRPA1, that TRPA1 activation may be an additional trigger for co-expressed calcium-dependent ion channels such as TRPM5, and that TRPM5 may amplify responses to TRPA1 ligands.

Caffeine activates mouse TRPA1 channels but suppresses human TRPA1 channels

Caffeine has various well-characterized pharmacological effects, but in mammals there are no known plasma membrane receptors or ion channels activated by caffeine. We observed that caffeine activates mouse transient receptor potential A1 (TRPA1) in heterologous expression systems by Ca(2+)(i) imaging and electrophysiological analyses. These responses to caffeine were confirmed in acutely dissociated dorsal root ganglion sensory neurons from WT mice, which are known to express TRPA1, but were not seen in neurons from TRPA1 KO mice. Expression of TRPA1 was detected immunohistochemically in nerve fibers and bundles in the mouse tongue. Moreover, WT mice, but not KO mice, showed a remarkable aversion to caffeine-containing water. These results demonstrate that mouse TRPA1 channels expressed in sensory neurons cause an aversion to drinking caffeine-containing water, suggesting they mediate the perception of caffeine. Finally, we observed that caffeine does not activate human TRPA1; instead, it suppresses its activity.