Transient Receptor Potential Ankyrin 1 Knockout Research Articles

FK506 (tacrolimus) causes pain sensation through the activation of transient receptor potential ankyrin 1 (TRPA1) channels.

FK506 (tacrolimus) is an immunosuppressant widely used as an ointment in the treatment of atopic dermatitis. However, local application of FK506 can evoke burning sensations in atopic dermatitis patients, and its mechanisms are unknown. In this study, we found that FK506 activates transient receptor potential ankyrin 1 (TRPA1) channels. In Ca2+-imaging experiments, increases in intracellular Ca2+ concentrations ([Ca2+]i) by FK506 were observed in HEK293T cells expressing hTRPA1 or hTRPM8. FK506-induced currents were observed in HEK293T cells expressing hTRPA1 or mTRPA1, but less or not at all in cells expressing hTRPV1 or hTRPM8 using a patch-clamp technique. FK506 also evoked single-channel opening of hTRPA1 in an inside-out configuration. FK506-induced [Ca2+]i increases were also observed in TRPA1-expressing mouse primary sensory neurons. Furthermore, injection of FK506 evoked licking or biting behaviors and these behaviors were almost abolished in TRPA1 knockout mice. These results indicate that FK506 might cause pain sensations through TRPA1 activation.

Abstract 292: TRPA1 Promoting Myofibroblast Differentiation Mediate Pressure Overload-Induced Cardiac Fibrosis

Background: Hypertensive ventricular remodeling is a common cause of heart failure. Activation and accumulation of cardiac fibroblasts is the key contributors to this progression. Our previous studies indicate that transient receptor potential ankyrin 1 (TRPA1), a Ca 2+ channel necessary and sufficient, play a prominent role in ventricular remodeling. However, the molecular mechanisms regulating remain poorly understood. Methods: We used TRPA1 agonists cinnamaldehyde (CA) pretreatment and TRPA1 knockout mice to understand the role of TRPA1 in ventricular remodeling of hypertensive heart. We also examine the mechanisms through gene transfection and in vitro experiments. Results: TRPA1 overexpression fully activated myofibroblast transformation, while fibroblasts lacking TRPA1 were refractory to transforming growth factor β (TGF-β) -induced transdifferentiation. TRPA1 knockout mice showed hypertensive ventricular remodeling reversal following pressure overload. We found that the TGF-β induced TRPA1 expression through calcineurin-NFAT-Dyrk1A signaling pathway via the TRPA1 promoter. Once induced, TRPA1 activates the Ca 2+ -responsive protein phosphatase calcineurin, which itself induced myofibroblast transdifferentiation. Moreover, inhibition of calcineurin prevented TRPA1-dependent transdifferentiation. Conclusion: Our study provides the first evidence that TRPA1 regulation in cardiac fibroblasts transformation in response to hypertensive stimulation. The results suggesting a comprehensive pathway for myofibroblast formation in conjunction with TGF-β, Calcineurin, NFAT and Dyrk1A. Furthermore, these data indicate that negative modulation of cardiac fibroblast TRPA1 may represent a therapeutic strategy against hypertensive cardiac remodeling.

The antipyretic effect of paracetamol occurs independent of transient receptor potential ankyrin 1-mediated hypothermia and is associated with prostaglandin inhibition in the brain.

The mode of action of paracetamol (acetaminophen), which is widely used for treating pain and fever, has remained obscure, but may involve several distinct mechanisms, including cyclooxygenase inhibition and transient receptor potential ankyrin 1 (TRPA1) channel activation, the latter being recently associated with paracetamol's propensity to elicit hypothermia at higher doses. Here, we examined whether the antipyretic effect of paracetamol was due to TRPA1 activation or cyclooxygenase inhibition. Treatment of wild-type and TRPA1 knockout mice rendered febrile by immune challenge with LPS with a dose of paracetamol that did not produce hypothermia (150 mg/kg) but is known to be analgetic, abolished fever in both genotypes. Paracetamol completely suppressed the LPS-induced elevation of prostaglandin E2 in the brain and also reduced the levels of several other prostanoids. The hypothermia induced by paracetamol was abolished in mice treated with the electrophile-scavenger N-acetyl cysteine. We conclude that paracetamol's antipyretic effect in mice is dependent on inhibition of cyclooxygenase activity, including the formation of pyrogenic prostaglandin E2, whereas paracetamol-induced hypothermia likely is mediated by the activation of TRPA1 by electrophilic metabolites of paracetamol, similar to its analgesic effect in some experimental paradigms.-Mirrasekhian, E., Nilsson, J. L. Å., Shionoya, K., Blomgren, A., Zygmunt, P. M., Engblom, D., Högestätt, E. D., Blomqvist, A. The antipyretic effect of paracetamol occurs independent of transient receptor potential ankyrin 1-mediated hypothermia and is associated with prostaglandin inhibition in the brain.

Endothelial TRPA1 Channels Are Activated by Hypoxia in Cerebral Arteries and Protect Against Ischemic Damage

Acute drops in cerebral perfusion can lead to rapid neuronal death, as seen during transient ischemic attacks and ischemic strokes. The extent of brain damage following hypoxia is linked to the ability of collateral blood vessels to adequately supply blood to the peri‐infarct region and thus prevent neuronal loss and infarct expansion. During hypoxia, reactive oxygen species (ROS) are increased and may activate the Ca2+‐permeable transient receptor potential ankyrin 1 (TRPA1) in endothelial cells, promoting vasodilation. Here, we tested the hypothesis that TRPA1 channels in endothelial cells are activated by hypoxia‐derived ROS, leading to cerebral artery dilation and reduced ischemic damage. Using a novel mouse model expressing the Ca2+ indicator GCaMP6f in endothelial cells (Tek:GCaMP6f), we show that 4‐hydroxynonenal (4‐HNE), a lipid peroxide, increased TRPA1 channel activity, detected as elementary Ca2+ influx events (TRPA1 sparklets). The TRPA1 inhibitor A967079 prevented 4‐HNE induction of TRPA1 sparklets in endothelial cells. Acute exposure of cerebral arteries to hypoxia (5% O2, 6% CO2, 89% N2) significantly increased 4‐HNE accumulation in cerebral arteries, which was linked to an increase in TRPA1 sparklets. Hypoxia induction of TRPA1 sparklets was blocked by the TRPA1 inhibitor A967079, and by PEGylated superoxide dismutase (PEG‐SOD), suggesting a role for intracellular generation of ROS. Incubation of arteries with the mitochondria‐targeted antioxidant mitoTEMPO prevented the hypoxia‐induced increase in TRPA1 sparklets, whereas extracellular SOD or the NADPH oxidase inhibitor apocynin had no effect. In pressure myography studies, hypoxia caused endothelium‐dependent dilation of isolated cerebral arteries, an effect that was prevented by the membrane‐permeable SOD mimetic tempol and by mitoTEMPO. Hypoxia‐induced dilation of cerebral arteries was partially inhibited by the TRPA1 antagonist A967079 and was reduced in arteries isolated from endothelial cell‐specific TRPA1 knockout mice (eTRPA1−/− mice). In vivo, loss of TRPA1 channels in endothelial cells increased infarct size following middle cerebral occlusion, a model of ischemic strokes. Reversely, TRPA1 activation with cinnamaldehyde (50 mg/kg) reduced infarct size in wildtype, but not eTRPA1−/−, mice. Together, these data suggest that TRPA1 channels in cerebral artery endothelial cells are early sensors of hypoxia and orchestrate a response that culminates with vasodilation, thereby reducing ischemic damage. Further, pharmacological activation of TRPA1 channels may be a putative therapeutic target to reduce cerebral ischemic damage.Support or Funding InformationSupported by the National Institutes of Health (R01HL091095 to SE) and the American Heart Association (15POST2472002 to PWP)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cerebral Capillary TRPA1 Channels Mediate Functional Hyperemia via Retrograde Conducted Vasodilation

Cerebral capillaries are the sites of nutrient exchange and removal of metabolic by‐products within the brain parenchyma. Recent studies showed that cerebral capillary endothelial cells (ECs) are central players in matching augmented neuronal metabolism to an increase in local blood perfusion, a process known as functional hyperemia. However, little is known about the molecular sensors present in capillary EC that orchestrate dilation of upstream parenchymal arterioles (PAs). We previously showed that the transient receptor potential ankyrin 1 (TRPA1) channel, a Ca2+‐permeable non‐selective cation channel, is present in ECs from cerebral arteries, but not in other peripheral vascular beds. We tested the hypothesis that TRPA1 is also present in cerebral capillary EC and its activation induces dilation of upstream PAs through a Ca2+‐dependent mechanism. Immunofluorescence staining of freshly isolated cerebral capillaries showed the presence of TRPA1, which was absent in EC‐specific TRPA1 knockout mice (eTRPA1−/−). Further, currents evoked by the TRPA1 agonist allyl isothyocyanate (AITC, 30 μM) in whole‐cell patch clamp electrophysiology experiments were blocked by the selective TRPA1 antagonist HC‐030031 (10 μM) and were absent from eTRPA1−/− mice. Using a recently‐described ex vivo capillary‐parenchymal arteriole preparation in which the capillary bed remains attached to pressurized PAs, we observed that picospritzing capillaries with AITC induced dilation of the upstream PA, an effect inhibited by HC‐030031 and absent in eTRPA1−/− mice. We then investigated the mechanisms underlying propagation of the vasodilatory signal from capillary to arteriolar EC. Using mice expressing the genetically‐encoded Ca2+ biosensor GCaMP6f in EC, we observed that picospritzing AITC onto capillary beds generated an intercellular Ca2+ wave that propagated from capillaries to the upstream PA. Pre‐incubation of preparations with the TRPA1 inhibitor HC‐030031 abolished the generation of Ca2+ waves. Moreover, the frequency of Ca2+ transients in arteriolar EC was increased immediately after dissipation of the Ca2+ wave, a process blocked by HC‐030031. Increases in arteriolar EC intracellular Ca2+ can activate intermediate and small conductance Ca2+‐activated K+ channels (IK and SK, respectively), leading to vasodilation. To test if this mechanism was responsible for PA dilation, preparations were incubated with the IK and SK channels inhibitors TRAM‐34 (1 μM) and apamin (1 μM), which blunted arteriolar dilation induced by picospritzing AITC onto capillaries. Somatosensory stimulation of the whisker barrel cortex, a model of functional hyperemia, caused a smaller increase in perfusion in eTRPA1−/− mice than in wildtype littermates. Taken together, these data suggest that functional TRPA1 channels are present in cerebral capillary EC and that activation of the channel elicits intercellular Ca2+ waves that propagate to upstream PAs, where it activates IK and SK channels to cause hyperpolarization and dilation, thus mediating functional hyperemia responses.Support or Funding InformationSupported by the National Institutes of Health (R01HL091095 to SE) and the American Heart Association (15POST2472002 to PWP)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

TRPA1 Acts in a Protective Manner in Imiquimod-Induced Psoriasiform Dermatitis in Mice

This study revealed the modulatory role of transient receptor potential ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1) cation channels in the Aldara-induced (5% imiquimod) murine psoriasis model using selective antagonists and genetically altered animals. We have also developed a refined localized model to enable internal controls and reduce systemic effects. Skin pathology was quantified by measuring skin thickness, scaling, blood flow, and analyzing dermal cellular infiltrate, whereas nocifensive behaviors were also observed. Cytokine gene expression profiles were measured exvivo. Psoriasiform dermatitis was significantly enhanced in TRPA1 knockout mice and with TRPA1 antagonist (A967079) treatment. By comparison, symptoms were decreased when TRPV1 function was inhibited. Imiquimod induced Ca2+ influx in TRPA1-, but not in TRPV1-expressing cell lines. Immunohistochemical studies revealed that CD4+ T helper cells express TRPA1 but not TRPV1 ion channels in mice skin. Compared with the TRPV1 knockout animals, additional elimination of the TRPA1 channels in the TRPV1/TRPA1 double knockout mice did not modify the outcome of the imiquimod-induced reaction, further supporting the dominant role of TRPV1 in the process. Our results suggest that the protective effects in psoriasiform dermatitis can be mediated by the activation of neuronal and nonneuronal TRPA1 receptors.

Role of Transient Receptor Potential Ankyrin 1 Ion Channel and Somatostatin sst4 Receptor in the Antinociceptive and Anti-inflammatory Effects of Sodium Polysulfide and Dimethyl Trisulfide

Transient receptor potential ankyrin 1 (TRPA1) non-selective ligand-gated cation channels are mostly expressed in primary sensory neurons. Polysulfides (POLYs) are Janus-faced substances interacting with numerous target proteins and associated with both protective and detrimental processes. Activation of TRPA1 in sensory neurons, consequent somatostatin (SOM) liberation and action on sst4 receptors have recently emerged as mediators of the antinociceptive effect of organic trisulfide dimethyl trisulfide (DMTS). In the frame of the present study, we set out to compare the participation of this mechanism in antinociceptive and anti-inflammatory effects of inorganic sodium POLY and DMTS in carrageenan-evoked hind-paw inflammation. Inflammation of murine hind paws was induced by intraplantar injection of carrageenan (3% in 30 µL saline). Animals were treated intraperitoneally with POLY (17 µmol/kg) or DMTS (250 µmol/kg) or their respective vehicles 30 min prior paw challenge and six times afterward every 60 min. Mechanical pain threshold and swelling of the paws were measured by dynamic plantar aesthesiometry and plethysmometry at 2, 4, and 6 h after initiation of inflammation. Myeloperoxidase (MPO) activity in the hind paws were detected 6 h after challenge by luminescent imaging. Mice genetically lacking TRPA1 ion channels, sst4 receptors and their wild-type counterparts were used to examine the participation of these proteins in POLY and DMTS effects. POLY counteracted carrageenan-evoked mechanical hyperalgesia in a TRPA1 and sst4 receptor-dependent manner. POLY did not influence paw swelling and MPO activity. DMTS ameliorated all examined inflammatory parameters. Mitigation of mechanical hyperalgesia and paw swelling by DMTS were mediated through sst4 receptors. These effects were present in TRPA1 knockout animals, too. DMTS inhibited MPO activity with no participation of the sensory neuron–SOM axis. While antinociceptive effects of POLY are transmitted by activation of peptidergic nerves via TRPA1, release of SOM and its effect on sst4 receptors, those of DMTS partially rely on SOM release triggered by other routes. SOM is responsible for the inhibition of paw swelling by DMTS, but TRPA1 does not contribute to its release. Modulation of MPO activity by DMTS is independent of TRPA1 and sst4.

Activation of Myofibroblast TRPA1 by Steroids and Pirfenidone Ameliorates Fibrosis in Experimental Crohn's Disease.

Background & AimsThe transient receptor potential ankyrin 1 (TRPA1) channel is highly expressed in the intestinal lamina propria, but its contribution to gut physiology/pathophysiology is unclear. Here, we evaluated the function of myofibroblast TRPA1 channels in intestinal remodeling.MethodsAn intestinal myofibroblast cell line (InMyoFibs) was stimulated by transforming growth factor-β1 to induce in vitro fibrosis. Trpa1 knockout mice were generated using the Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system. A murine chronic colitis model was established by weekly intrarectal trinitrobenzene sulfonic acid (TNBS) administration. Samples from the intestines of Crohn’s disease (CD) patients were used for pathologic staining and quantitative analyses.ResultsIn InMyoFibs, TRPA1 showed the highest expression among TRP family members. In TNBS chronic colitis model mice, the extents of inflammation and fibrotic changes were more prominent in TRPA1-/- knockout than in wild-type mice. One-week enema administration of prednisolone suppressed fibrotic lesions in wild-type mice, but not in TRPA1 knockout mice. Steroids and pirfenidone induced Ca2+ influx in InMyoFibs, which was antagonized by the selective TRPA1 channel blocker HC-030031. Steroids and pirfenidone counteracted transforming growth factor-β1–induced expression of heat shock protein 47, type 1 collagen, and α-smooth muscle actin, and reduced Smad-2 phosphorylation and myocardin expression in InMyoFibs. In stenotic intestinal regions of CD patients, TRPA1 expression was increased significantly. TRPA1/heat shock protein 47 double-positive cells accumulated in the stenotic intestinal regions of both CD patients and TNBS-treated mice.ConclusionsTRPA1, in addition to its anti-inflammatory actions, may protect against intestinal fibrosis, thus being a novel therapeutic target for highly incurable inflammatory/fibrotic disorders.

Methylglyoxal Requires AC1 and TRPA1 to Produce Pain and Spinal Neuron Activation.

Methylglyoxal (MG) is a metabolite of glucose that may contribute to peripheral neuropathy and pain in diabetic patients. MG increases intracellular calcium in sensory neurons and produces behavioral nociception via the cation channel transient receptor potential ankyrin 1 (TRPA1). However, rigorous characterization of an animal model of methylglyoxal-evoked pain is needed, including testing whether methylglyoxal promotes negative pain affect. Furthermore, it remains unknown whether methylglyoxal is sufficient to activate neurons in the spinal cord dorsal horn, whether this requires TRPA1, and if the calcium-sensitive adenylyl cyclase 1 isoform (AC1) contributes to MG-evoked pain. We administered intraplantar methylglyoxal and then evaluated immunohistochemical phosphorylation of extracellular signal-regulated kinase (p-ERK) and multiple pain-like behaviors in wild-type rats and mice and after disruption of either TRPA1 or AC1. Methylglyoxal produced conditioned place avoidance (CPA) (a measure of affective pain), dose-dependent licking and lifting nociceptive behaviors, hyperalgesia to heat and mechanical stimulation, and p-ERK in the spinal cord dorsal horn. TRPA1 knockout or intrathecal administration of a TRPA1 antagonist (HC030031) attenuated methylglyoxal-evoked p-ERK, nociception, and hyperalgesia. AC1 knockout abolished hyperalgesia but not nociceptive behaviors. These results indicate that intraplantar administration of methylglyoxal recapitulates multiple signs of painful diabetic neuropathy found in animal models of or patients with diabetes, including the activation of spinal nociresponsive neurons and the potential involvement of a TRPA1-AC1 sensitization mechanism. We conclude that administration of MG is a valuable model for investigating both peripheral and central components of a MG-TRPA1-AC1 pathway that contribute to painful diabetic neuropathy.

\u03b2-Eudesmol, an oxygenized sesquiterpene, stimulates appetite via TRPA1 and the autonomic nervous system

Transient receptor potential ankyrin 1 (TRPA1) is a calcium-permeable non-selective cation channel, which is activated by various noxious or irritant substances in nature. TRPA1 activators have been generally recognized as noxious, however, foods and beverages containing TRPA1 activators are preferably consumed; the reasons for this discrepancy are not well understood. We demonstrate that TRPA1 is involved in the stimulatory appetite control mechanism. β-Eudesmol is an oxygenated sesquiterpene contained in medicinal or edible plants which activates TRPA1. Oral administration of β-eudesmol brought significant increments in food intake in rats and elevated plasma ghrelin levels. Gastric vagal nerve activity (GVNA) has been reported to affect feeding behavior. In vivo electrophysiological measurement of GVNA revealed that oral-ingestion of β-eudesmol significantly increased GVNA. This GVNA elevation was eliminated by TRPA1 inhibitor (HC-030031) treatment prior to β-eudesmol administration. The physiological effects of β-eudesmol, for example, incremental increase in food intake, ghrelin elevation and activation of GVNA, were significantly reduced in TRPA1 knockout rats. Our results indicated that β-eudesmol stimulates an increase in appetite through TRPA1, and suggests why TRPA1 activator containing foods and beverages are preferably consumed.

Nasal TRPA1 mediates irritant-induced bradypnea in mice.

Transient receptor potential ankyrin 1 (TRPA1), a member of the TRP superfamily, exists in sensory neurons such as trigeminal neurons innervating the nasal cavity and vagal neurons innervating the trachea and the lung. Although TRPA1 has been proposed as an irritant receptor that, when stimulated, triggers bradypnea, precise locations of the receptors responsible have not been elucidated. Here, we examined the relative importance of TRPA1 located in the upper airway (nasal) and the lower airway (trachea/lungs) in urethane‐anesthetized mice. To stimulate the upper and lower airways separately, two cannulas were inserted through a hole made in the trachea just caudal to the thyroid cartilage, one into the nasal cavity and the second into the lower trachea. A vapor of one of the TRPA1‐agonists, allyl isothiocyanate (AITC), was introduced by placing a piece of cotton paper soaked with AITC solution into the airline. AITC decreased the respiratory frequency when applied to the upper airway (ca −30%) but not to the lower airway (ca −5%). No response was observed in TRPA1 knockout mice. Contribution of the olfactory nerve seemed minimal because olfactory bulbectomized wild‐type mice showed a similar response to that of the intact mice. AITC‐induced bradypnea seemed to be mediated, at least in part, by the trigeminal nerve because trigeminal ganglion neurons were activated by AITC as revealed by an increase in the phosphorylated form of extracellular signal‐regulated kinase in the neurons. These data clearly show that trigeminal TRPA1 in the nasal cavity play an essential role in irritant‐induced bradypnea.

Transient receptor potential ankyrin 1 (TRPA1) receptor is involved in chronic arthritis: in vivo study using TRPA1-deficient mice.

BackgroundThe transient receptor potential ankyrin 1 (TRPA1) is a calcium-permeable cation channel that is expressed on capsaicin-sensitive sensory neurons, endothelial and inflammatory cells. It is activated by a variety of inflammatory mediators, such as methylglyoxal, formaldehyde and hydrogen sulphide. Since only few data are available about the role of TRPA1 in arthritis and related pain, we investigated its involvement in inflammation models of different mechanisms.MethodsChronic arthritis was induced by complete Freund’s adjuvant (CFA), knee osteoarthritis by monosodium iodoacetate (MIA) in TRPA1 knockout (KO) mice and C57Bl/6 wildtype mice. For comparison, carrageenan- and CFA-evoked acute paw and knee inflammatory changes were investigated. Thermonociception was determined on a hot plate, cold tolerance in icy water, mechanonociception by aesthesiometry, paw volume by plethysmometry, knee diameter by micrometry, weight distribution with incapacitance tester, neutrophil myeloperoxidase activity and vascular leakage by in vivo optical imaging, and histopathological alterations by semiquantitative scoring.ResultsCFA-induced chronic mechanical hypersensitivity, tibiotarsal joint swelling and histopathological alterations, as well as myeloperoxidase activity in the early phase (day 2), and vascular leakage in the later stage (day 7), were significantly reduced in TRPA1 KO mice. Heat and cold sensitivities did not change in this model. Although in TRPA1 KO animals MIA-evoked knee swelling and histopathological destruction were not altered, hypersensitivity and impaired weight bearing on the osteoarthritic limb were significantly decreased. In contrast, carrageenan- and CFA-induced acute inflammation and pain behaviours were not modified by TRPA1 deletion.ConclusionsTRPA1 has an important role in chronic arthritis/osteoarthritis and related pain behaviours in the mouse. Therefore, it might be a promising target for novel analgesic/anti-inflammatory drugs.

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

Endothelial Cell TRPA1 Channel Activity Delays the Onset of Hypertension‐Associated Hemorrhagic Stroke

The Ca2+-permeable transient receptor potential ankyrin 1 (TRPA1) channel is present in cerebral artery endothelial cells and mediates endothelium-dependent dilation in response to oxidized lipid metabolites. Prior studies have shown that oxidative degradation of membrane lipids is increased in the vascular wall during hypertension. Therefore, we hypothesized that TRPA1 activity is enhanced in cerebral artery endothelial cells during hypertension and that augmented activity is protective against stroke. Hypertension was induced in control and endothelial cell specific TRPA1 knockout mice (eTRPA1-/-) by infusion of angiotensin II (1200 ng/kg/min, s.c.) and high salt (8%) diet. After two weeks of this treatment, the nitric oxide synthase inhibitor L-NAME (120 mg/kg/day) was added to the drinking water to further increase mean arterial pressure (MAP). MAP was monitored by radiotelemetery and did not differ between groups under any conditions. All mice suffered fatal stroke. The survival time of eTRPA1-/- mice (6-22 days) was significantly less than that of control (3-52 days) littermates (n = 19-23). Histopathology revealed that eTRPA1-/- mice developed more intracerebral hemorrhage (ICH) lesions than control mice (75 ± 1 total lesions per eTRPA1-/- mouse vs. 50 ± 9 lesions per control mouse, n = 3-4 per group). However, the volume of each lesion was smaller in eTRPA1-/- mice (0.03 ± 0.01 mm3 for eTRPA1-/- vs. 0.12 ± 0.04 mm3 for control, n = 3-4). Together these data provide evidence that TRPA1 channels present in the cerebral endothelium are protective against hemorrhagic stroke associated with hypertension. Supported by HL091905 and 11SDG7360050.

Investigating the potential role of TRPA1 in locomotion and cardiovascular control during hypertension.

Radiotelemetry was used to investigate the in vivo cardiovascular and activity phenotype of both TRPA1 (transient receptor potential ankyrin 1) wild-type (WT) and TRPA1 knockout (KO) mice. After baseline recording, experimental hypertension was induced using angiotensin II infusion (1.1 mg−1 kg−1 a day, for 14 days). TRPA1 WT and KO mice showed similar morphological and functional cardiovascular parameters, including similar basal blood pressure (BP), heart rate, size, and function. Similar hypertension was also displayed in response to angiotensin II (156 ± 7 and 165 ± 11 mmHg, systolic BP ± SEM, n = 5–6). TRPA1 KO mice showed increased hypertensive hypertrophy (heart weight:tibia length: 7.3 ± 1.6 mg mm−1 vs. 8.8 ± 1.7 mg mm−1) and presented with blunted interleukin 6 (IL-6) production compared with hypertensive WT mice (151 ± 24 vs. 89 ± 16 pg mL−1). TRPA1 expression in dorsal root ganglion (DRG) neurones was upregulated during hypertension (163% of baseline expression). Investigations utilizing the TRPA1 agonist cinnamaldehyde (CA) on mesenteric arterioles isolated from näive mice suggested a lack of TRPA1-dependent vasoreactivity in this vascular bed; a site with notable ability to alter total peripheral resistance. However, mesenteric arterioles isolated from TRPA1 KO hypertensive mice displayed significantly reduced ability to relax in response to nitric oxide (NO) (P < 0.05). Unexpectedly, naïve TRPA1 KO mice also displayed physical hyperactivity traits at baseline, which was exacerbated during hypertension. In conclusion, our study provides a novel cardiovascular characterization of TRPA1 KO mice in a model of hypertension. Results suggest that TRPA1 has a limited role in global cardiovascular control, but we demonstrate an unexpected capacity for TRPA1 to regulate physical activity.

TRPA1 detects environmental chemicals and induces avoidance behavior and arousal from sleep

Detecting threats and escaping before serious confrontations are important for animals to avoid danger and death. Transient receptor potential ankyrin 1 (TRPA1), a member of the TRP superfamily, is expressed in a subset of sensory neurons and mediates nociception evoked by pungent chemicals. Using behavioral testing, we found that TRPA1 knockout mice failed to avoid entering a chamber filled with vapor of formalin, allyl isothiocyanate, and acrolein. The avoidance behavior was blocked by nasal but not subcutaneous administration of a blocker to TRPA1. We also found that TRPA1 knockout mice did not wake when exposed to formalin during sleep. Additionally, the spinal trigeminal nucleus, the first relay neurons of the trigeminal system, showed massive expression of c-Fos after a brief (3 min) exposure to formalin vapor. TRPA1 seems to be a sentinel for environmental chemicals and induces avoidance behaviors and waking by way of the trigeminal system.

Avoidance of environmental gas irritants mediated by TRPA1

Transient receptor potential ankyrin 1 (TRPA1), a member of the TRP superfamily, is expressed in a subset of sensory neurons such as trigeminal and dorsal root ganglia and implicated as a mediator of nociception evoked by diverse peripheral stimuli including pungent chemicals. We hypothesized that TRPA1 would be involved in detecting environmental chemicals to avoid taking in such a threat. To test our hypothesis, we did place preference test using TRPA1 knockout (KO) mice and age matched wild‐type (WT) mice. The mice were first allowed to freely explore a homemade apparatus that consists of four chambers and the connecting tubes. The number of entry times and the amount of time spent in each chamber were recorded. None of the animals had initial bias for either chamber. Then, each chamber was randomly assigned to a chamber in which a piece of cotton paper soaked with a test solution was placed. We tested a TRPA1 agonist, formaldehyde. During a test period of 20 min, WT mice never tried to enter the chamber with formaldehyde. KO mice entered the chamber without hesitation and even stayed there. This result indicates that WT mice detect and recognize formaldehyde as a dangerous substance via TRPA1.

TRPA1 is an environmental gas alarm

Transient receptor potential ankyrin 1 (TRPA1), a member of the TRP super family, is expressed in a subset of sensory neurons in the trigeminal, nodose, and dorsal root ganglia and implicated as a mediator of nociception evoked by diverse peripheral stimuli including noxious cold and pungent chemicals. We hypothesized that TRPA1 would be involved in detecting environmental chemicals to avoid taking in such a threat. To test our hypothesis, we did place preference test using TRPA1 knockout (KO) mice and age matched wild-type (WT) mice.

Evidence for the pathophysiological relevance of TRPA1 receptors in the cardiovascular system in vivo

The aim of the study is to investigate transient receptor potential ankyrin 1 (TRPA1)-induced responses in the vasculature and on blood pressure and heart rate (HR), in response to TRPA1 agonists using wild-type (WT) and TRPA1 knockout (KO) mice. TRPA1 agonists allyl isothiocyanate and cinnamaldehyde (CA) significantly increased blood flow in the skin of anaesthetized WT, but not in TRPA1 KO mice. CA also induced TRPA1-dependent relaxation of mesenteric arteries. Intravenously injected CA induced a transient hypotensive response accompanied by decreased HR that was, depending on genotype and dose, followed by a more sustained dose-dependent pressor response (10-320 micromol/kg). CA (80 micromol/kg) induced a depressor response that was significantly less in TRPA1 KO mice, with minimal pressor effects. The pressor response of a higher CA dose (320 micromol/kg) was observed in WT but not in TRPA1 KO mice, indicating involvement of TRPA1. Experiments using TRP vanilloid 1 (TRPV1) KO and calcitonin gene-related peptide (CGRP) KO mice provided little evidence for the involvement of TRPV1 or CGRP, nor did blocking substance P receptors affect responses. However, the cholinergic antagonist atropine sulphate (5 mg/kg) significantly inhibited the depressor response and slowed HR with CA (80 micromol/kg), but had no effect on pressor responses. The pressor response remained unaffected, even in the presence of the ganglion blocker hexamethonium bromide (1 mg/kg). The alpha-adrenergic blocker prazosin hydrochloride (1 mg/kg) significantly inhibited both components, but not slowed HR. TRPA1 is involved in mediating vasodilation. TRPA1 can also influence changes in blood pressure of possible relevance to autonomic system reflexes and potentially to vasovagal/neurocardiogenic syncope disorders.

Species-Specific Pharmacology of Trichloro(Sulfanyl)ethyl Benzamides as Transient Receptor Potential Ankyrin 1 (TRPA1) Antagonists

Agonists of TRPA1 such as mustard oil and its key component AITC cause pain and neurogenic inflammation in humans and pain behavior in rodents. TRPA1 is activated by numerous reactive compounds making it a sensor for reactive compounds in the body. Failure of AITC, formalin and other reactive compounds to trigger pain behavior in TRPA1 knockout mice, as well as the ability of TRPA1 antisense to alleviate cold hyperalgesia after spinal nerve ligation, suggest that TRPA1 is a potential target for novel analgesic agents. Here, we have characterized CHO cells expressing human and rat TRPA1 driven by an inducible promoter. As reported previously, both human and rat TRPA1 are activated by AITC and inhibited by ruthenium red. We have also characterized noxious cold response of these cell lines and show that noxious cold activates both human and rat TRPA1. Further, we have used CHO cells expressing human TRPA1 to screen a small molecule compound library and discovered that 'trichloro(sulfanyl)ethyl benzamides' (AMG2504, AMG5445, AMG7160 and AMG9090) act as potent antagonists of human TRPA1 activated by AITC and noxious cold. However, trichloro(sulfanyl)ethyl benzamides' (TCEB compounds) displayed differential pharmacology at rat TRPA1. AMG2504 and AMG7160 marginally inhibited rat TRPA1 activation by AITC, whereas AMG5445 and AMG9090 acted as partial agonists. In summary, we conclude that both human and rat TRPA1 channels show similar AITC and noxious cold activation profiles, but TCEB compounds display species-specific differential pharmacology at TRPA1.