Transient Receptor Potential Ankyrin 1 Agonist Research Articles

Phosphorylation of the Transient Receptor Potential Ankyrin 1 by Cyclin-dependent Kinase 5 affects Chemo-nociception

Cyclin-dependent kinase 5 (Cdk5) is a key neuronal kinase that is upregulated during inflammation, and can subsequently modulate sensitivity to nociceptive stimuli. We conducted an in silico screen for Cdk5 phosphorylation sites within proteins whose expression was enriched in nociceptors and identified the chemo-responsive ion channel Transient Receptor Potential Ankyrin 1 (TRPA1) as a possible Cdk5 substrate. Immunoprecipitated full length TRPA1 was shown to be phosphorylated by Cdk5 and this interaction was blocked by TFP5, an inhibitor that prevents activation of Cdk5. In vitro peptide-based kinase assay revealed that four of six TRPA1 Cdk5 consensus sites acted as substrates for Cdk5, and modeling of the ankyrin repeats disclosed that phosphorylation would occur at characteristic pockets within the (T/S)PLH motifs. Calcium imaging of trigeminal ganglion neurons from genetically engineered mice overexpressing or lacking the Cdk5 activator p35 displayed increased or decreased responsiveness, respectively, to stimulation with the TRPA1 agonist allylisothiocyanate (AITC). AITC-induced chemo-nociceptive behavior was also heightened in vivo in mice overexpressing p35 while being reduced in p35 knockout mice. Our findings demonstrate that TRPA1 is a substrate of Cdk5 and that Cdk5 activity is also able to modulate TRPA1 agonist-induced calcium influx and chemo-nociceptive behavioral responses.

IL-1β augments H2S-induced increase in intracellular Ca2+ through polysulfides generated from H2S/NO interaction

H2S has excitatory and inhibitory effects on Ca2+ signals via transient receptor potential ankyrin 1 (TRPA1) and ATP-sensitive K+ channels, respectively. H2S converts intracellularly to polysulfides, which are more potent agonists for TRPA1 than H2S. Under inflammatory conditions, changes in the expression and activity of these H2S target channels and/or the conversion of H2S to polysulfides may modulate H2S effects. Effects of proinflammatory cytokines on H2S-induced Ca2+ signals and polysulfide production in RIN14B cells were examined using fluorescence imaging with fura-2 and SSP4, respectively. Na2S, a H2S donor, induced 1) the inhibition of spontaneous Ca2+ signals, 2) inhibition followed by [Ca2+]i increase, and 3) rapid [Ca2+]i increase without inhibition in 50% (23/46), 22% (10/46), and 17% (8/46) of cells tested, respectively. IL-1β augmented H2S-induced [Ca2+]i increases, which were inhibited by TRPA1 and voltage-dependent L-type Ca2+ channel blockers. However, IL-1β treatment did not affect [Ca2+]i increases evoked by a TRPA1 agonist or high concentration of KCl. Na2S increased intracellular polysulfide levels, which were enhanced by IL-1β treatment. A NOS inhibitor suppressed the increased polysulfide production and [Ca2+]i increase in IL-1β-treated cells. These results suggest that IL-1β augments H2S-induced [Ca2+]i increases via the conversion of H2S to polysulfides through NO synthesis, but not via changes in the activity and expression of target channels. Polysulfides may play an important role in the effects of H2S during inflammation.

TRPA1 sensitization during diabetic vascular impairment contributes to cold hypersensitivity in a mouse model of painful diabetic peripheral neuropathy.

BackgroundDiabetic peripheral neuropathy is a common long-term complication of diabetes. Accumulating evidence suggests that vascular impairment plays important roles in the pathogenesis of diabetic peripheral neuropathy, while the mechanism remains unclear. We recently reported that transient receptor potential ankyrin 1 (TRPA1) is sensitized by hypoxia, which can contribute to cold hypersensitivity. In this study, we investigated the involvement of TRPA1 and vascular impairment in painful diabetic peripheral neuropathy using streptozotocin-induced diabetic model mice.ResultsStreptozotocin-induced diabetic model mice showed mechanical and cold hypersensitivity with a peak at two weeks after the streptozotocin administration, which were likely to be paralleled with the decrease in the skin blood flow of the hindpaw. Streptozotocin-induced cold hypersensitivity was significantly inhibited by an antagonist HC-030031 (100 mg/kg) or deficiency for TRPA1, whereas mechanical hypersensitivity was unaltered. Consistent with these results, the nocifensive behaviors evoked by an intraplantar injection of the TRPA1 agonist allyl isothiocyanate (AITC) were enhanced two weeks after the streptozotocin administration. Both streptozotocin-induced cold hypersensitivity and the enhanced AITC-evoked nocifensive behaviors were significantly inhibited by a vasodilator, tadalafil (10 mg/kg), with recovery of the decreased skin blood flow. Similarly, in a mouse model of hindlimb ischemia induced by the ligation of the external iliac artery, AITC-evoked nocifensive behaviors were significantly enhanced three and seven days after the ischemic operation, whereas mechanical hypersensitivity was unaltered in TRPA1-knockout mice. However, no difference was observed between wild-type and TRPA1-knockout mice in the hyposensitivity for current or mechanical stimulation or the deceased density of intraepidermal nerve fibers eight weeks after the streptozotocin administration.ConclusionThese results suggest that TRPA1 sensitization during diabetic vascular impairment causes cold, but not mechanical, hypersensitivity in the early painful phase of diabetic peripheral neuropathy. However, TRPA1 may play little or no role in the progression of diabetic peripheral neuropathy.

Abstract 003: Role of Transient Receptor Potential Ankyrin 1 in Monocyte Activation and Hypertensive Kidney Injury

It has been shown that monocyte activation and infiltration occur during the development of hypertensive kidney disease, a condition newly recognized as an inflammatory disorder. Indeed, monocytosis is linked to chronic kidney disease. Transient receptor potential ankyrin 1 (TRPA1), a Ca 2+ -permeable non-selective cation channel, has been shown to be an oxidative stress sensor that regulates neurogenic inflammation due to its abundant expression in sensory nerves. With the use of qPCR and immunostaining, we identified that the TRPA1 is also expressed in human and murine monocytes. The gene expression of TRPA1 is decreased in peripheral monocytes in hypertensive patients compared to normotensive subjects (-6-fold, p<0.05) and in kidney tissues in angiotensin II (1000ng/kg/min, s.c., 14 days)-induced hypertensive compared to control mice (-9-fold, p<0.01). The differentiation of human monocytic leukemia cells (THP-1) into macrophages induced by phorbol 12-myristate 13-acetate (PMA, 100nM, 48h) was accompanied with decreases in TRPA1 expression (-8-fold, p<0.05) and increases in pro-inflammatory cytokines, including interleukin (IL)-1β (+705-fold, p<0.01), monocyte chemotactic protein (MCP)-1 (+16-fold, p<0.01), and CD36 (+7-fold, p<0.01). Treatment with supercinnamaldehyde (SCA), a TRPA1 agonist, decreased the expression of IL-1β (-12-fold, p<0.01), MCP-1 (-3.5-fold, p<0.01), and CD36 (-2-fold, p<0.05) in THP-1-derived macrophages, whereas these effects were abolished by pre-treatment with the selective TRPA1 antagonist, HC-030031 (HC). Additionally, HC promoted the proliferation of THP-1 cells while SCA suppressed THP-1 cell growth and variability. SCA induced the apoptosis of THP-1 cells (Annexin V positive cells: 8.5±1.4% vs. 30.1±2.4%, p<0.01), which can be blocked by HC (30.1±2.4% vs. 15.6±1.9%, p<0.01). These results show that TRPA1 activation inhibits monocyte activation and differentiation, and that suppressed expression of TRPA1 occurs in monocytes and kidneys in hypertensive patients and animals, respectively. These findings indicate that impaired expression and function of TRPA1 may contribute to monocytosis and pro-inflammatory state during hypertension, leading to hypertension related kidney injury.

TRPA1 activation in a human sensory neuronal model: relevance to cough hypersensitivity?

The cough reflex becomes hyperresponsive in acute and chronic respiratory diseases, but understanding the underlying mechanism is hampered by difficulty accessing human tissue containing both nerve endings and neuronal cell bodies. We refined an adult stem cell sensory neuronal model to overcome the limited availability of human neurones and applied the model to study transient receptor potential ankyrin 1 (TRPA1) channel expression and activation.Human dental pulp stem cells (hDPSCs) were differentiated towards a neuronal phenotype, termed peripheral neuronal equivalents (PNEs). Using molecular and immunohistochemical techniques, together with Ca2+ microfluorimetry and whole cell patch clamping, we investigated roles for nerve growth factor (NGF) and the viral mimic poly I:C in TRPA1 activation.PNEs exhibited morphological, molecular and functional characteristics of sensory neurons and expressed functional TRPA1 channels. PNE treatment with NGF for 20 min generated significantly larger inward and outward currents compared to untreated PNEs in response to the TRPA1 agonist cinnamaldehyde (p<0.05). PNE treatment with poly I:C caused similar transient heightened responses to TRPA1 activation compared to untreated cells.Using the PNE neuronal model we observed both NGF and poly I:C mediated sensory neuronal hyperresponsiveness, representing potential neuro-inflammatory mechanisms associated with heightened nociceptive responses recognised in cough hypersensitivity syndrome.

Membrane translocation of transient receptor potential ankyrin 1 induced by inflammatory cytokines in lung cancer cells

Transient receptor potential ankyrin 1 (TRPA1) is known as one of the nociceptors expressed in sensory neurons. It also plays a role in non-neural cells in inflammatory sites. However, the regulatory mechanisms for the reactivity of TRPA1 in these cells under inflammatory conditions are not clear. To clarify these mechanisms, we examined the effects of inflammatory cytokines (interleukin [IL]-1α, IL-1β and tumor necrosis factor α [TNFα]) on TRPA1 reactivity and expression in the endogenously TRPA1-expressing lung tumor cell line A549. Treatment with IL-1α, but not IL-1β or TNFα, increased the number of cells responding to allyl isothiocyanate, a TRPA1 agonist, in a dose- and time-dependent manner. The IL-1α-induced increase of TRPA1 responsiveness was inhibited by an extracellular-regulated kinase (Erk) inhibitor (PD98059) but not by inhibitors of c-Jun kinase, p38 mitogen-activated protein kinase or phosphatidylinositol-3 kinase. Phosphorylation of Erk gradually increased at 24 h after its transient induction in cells treated with IL-1α. IL-1α increased the TRPA1 levels on biotinylated cell surface proteins. These results suggest that IL-1α enhances the translocation of TRPA1 to the plasma membrane via the activation of Erk in A549. TRPA1 may have a pathophysiological role in non-neural lung cells under inflammatory conditions.

TRPA1 Channels Modify TRPV1-Mediated Current Responses in Dorsal Root Ganglion Neurons.

The transient receptor potential vanilloid 1 (TRPV1) channel is highly expressed in a subset of sensory neurons in the dorsal root ganglia (DRG) and trigeminal ganglia of experimental animals, responsible for nociception. Many researches have revealed that some TRPV1-positive neurons co-express the transient receptor potential ankyrin 1 (TRPA1) channel whose activities are closely modulated by TRPV1 channel. However, it is less investigated whether the activities of TRPV1 channel are modulated by the presence of TRPA1 channel in primary sensory neurons. This study clarified the difference in electrophysiological responses induced by TRPV1 channel activation between TRPA1-positive and TRPA1-negative DRG. TRPV1 and TRPA1 channel activations were evoked by capsaicin (1 μM), a TRPV1 agonist, and allyl isothiocyanate (AITC; 500 μM), a TRPA1 agonist, respectively. Capsaicin perfusion for 15 s caused a large inward current without a desensitization phase at a membrane potential of −70 mV in AITC-insensitive DRG (current density; 29.6 ± 5.6 pA/pF, time constant of decay; 12.8 ± 1.8 s). The capsaicin-induced currents in AITC-sensitive DRG had a small current density (12.7 ± 2.9 pA/pF) with a large time constant of decay (24.3 ± 5.4 s). In calcium imaging with Fura-2, the peak response by capsaicin was small and duration reaching the peak response was long in AITC-sensitive neurons. These electrophysiological differences were completely eliminated by HC-030031, a TRPA1 antagonist, in an extracellular solution or 10 mM EGTA, a Ca2+ chelator, in an internal solution. Capsaicin perfusion for 120 s desensitized the inward currents after a transient peak. The decay during capsaicin perfusion was notably slow in AITC-sensitive DRG; ratio of capsaicin-induced current 60 s after the treatment per the peak current in AITC-sensitive neurons (78 ± 9%) was larger than that in AITC-insensitive neurons (48 ± 5%). The capsaicin-induced current in the desensitization phase was attenuated by HC-030031 in AITC-insensitive DRG. These results indicate that (1) TRPV1-mediated currents in TRPA1-positive neurons characterize small current densities with slow decay, which is caused by TRPA1 channel activities and intracellular Ca2+ mobilization and (2) desensitization of TRPV1-mediated current in TRPA1-positive neurons is apparently slow, due to appending TRPA1-mediated current.

Discrepancy between TRPA1 activation by reversible and irreversible electrophiles suggests involvement of cytosolic cofactors other than polyphosphates.

Transient Receptor Potential Ankyrin 1 (TRPA1) is a non‐selective cation channel that is expressed in a subset of nociceptive afferent sensory nerves. When activated, TRPA1 can evoke nociceptive responses (e.g. pain and defensive reflexes), as well as peripheral neurogenic inflammation. TRPA1 is activated by a host of endogenous (inflammatory mediators) and exogenous (pollutants, food) agonists. Many TRPA1 agonists are electrophilic compounds that covalently modify reactive cysteine residues on TRPA1, leading to activation of the channel. Electrophiles can bind through reversible or irreversible reactions based on the structure of the compound. Here, patch clamp techniques and calcium imaging have been used to characterize human TRPA1 (hTRPA1) channel activity (expressed in HEK293 cells) in the presence of reversible and irreversible electrophiles. Calcium imaging shows that both reversible electrophiles (e.g. allylisothiocyanate [AITC]) and irreversible electrophiles (e.g. iodoacetamide [IA] and N‐ethylmaleimide [NEM]) rapidly induce TRPA1 activation. In contrast, our patch clamp studies show a discrepancy between irreversible and reversible electrophilic TRPA1 activation. Whole cell patch clamp studies exhibit a reduction in the kinetics and magnitude of TRPA1 activation by IA and NEM compared to AITC. Furthermore, single channel patch clamp experiments display reduced open probability with IA and NEM compared to AITC. Both whole cell patch and excised patch clamp recordings were performed in the presence of 5mM polyphosphates. In preliminary gramicidin perforated patch recordings there was no difference in NEM and AITC‐evoked TRPA1 activation. Thus in intact cellular assays (Ca2+ imaging and gramicidin‐perforated patch) there is little difference in TRPA1 activation by reversible and irreversible electrophiles, whereas in dialyzed cellular assays (whole cell patch and excised patch) irreversible electrophiles evoked reduced TRPA1 activation compared to reversible electrophiles. The data suggests that cytosolic factors other than polyphosphates are important for TRPA1 activation by irreversible electrophilic ligands.Support or Funding InformationNHLBI R01 (HL119802) and Supplement (HL119802‐S1)

Structural Insight into the Molecular Mechanism of TRPA1 Inhibition and Activation

Pain, while serving the beneficial function of provoking our attention to dangerous situations, is an unpleasant sensory and emotional experience. Transient Receptor Potential Ankyrin 1 (TRPA1) is a member of the transient receptor potential (TRP) cation channel family and is localized in nerve fibers called “nociceptors” where it plays a key role in the transduction of chemical, inflammatory and neuropathic pain signals from the periphery to the brain.TRPA1 is a non-selective ion channel, which is activated by a variety of structurally diverse compounds. TRPA1 agonists are exogenous electrophilic compounds and endogenous thiol-reactive inflammatory mediators, all of which target and activate the channel via a cluster of cysteine residues at the N-terminus. Activation is clearly dependent on a thiol-reactive moiety, thus explaining the structural diversity of this group. Although near-atomic resolution structures of TRPA1 were resolved recently by cryo-EM, both in the presence of agonist and antagonists, detailed mechanisms of channel activation and inhibition by these modulators could not be determined.To determine the structural details of TRPA1 conformation upon agonist and antagonist binding, we used limited proteolysis and mass spectrometry. Our study indicates that conformational rearrangements of the N-terminus ankyrin repeats and the linker region between C-terminus and the transmembrane domain contribute to the channel modulation.

Modulation by Phenolic Compounds Provides Novel Insight into the Mechanisms of TRPA1 Activation

Transient receptor potential ankyrin 1 (TRPA1) is a cation channel activated by a large number of noxious chemicals found in many plants, foods, cosmetics, drugs and pollutants. These substances belong to different chemical classes with the specific functional groups responsible for the channel activation. Numerous TRPA1 agonists are highly reactive electrophiles, and have ability to bind and modify thiol groups in the cytosolic part of the channel. Another group of compounds contains a large number of non-electrophiles, such as menthol, clotrimazole and nicotine, which are unable to covalently modify TRPA1. Non-electrophilic agonists have extremely different chemical structures and functions, so it seems very unlikely that a single channel structure or binding pocket could accommodate direct binding of so many diverse compounds. However, one common feature of many of these species is the ability to partition into the plasma membrane, which may in turn induce mechanical perturbations. Using intracellular calcium measurements and patch-clamp recordings we found that non-electrophilic phenol derivatives activate mouse TRPA1 and that this effect is prevented by the specific channel inhibitor HC030031. The analysis of the effects of different phenol derivatives revealed a reduction of the EC50 for TRPA1 activation with increasing length of carbon side chain in positions orto, meta and para on the phenol ring. The lengthening of the carbonyl side chain correlates also with the increase in octanol/water partition coefficient and the ability of the compounds to insert into cellular membranes. In fluorescence spectroscopy and imaging experiments with the fluorophores laurdan and 1,6-diphenyl-1,3,5-hexatriene (DPH) we found that phenol derivatives modulate membrane properties. We propose that activation of TRPA1 by non-electrophilic compounds may arise from the induction of changes in membrane properties and suggest that chemosensation may result from primary mechanosensory mechanisms.

Environmental toxin acrolein alters levels of endogenous lipids, including TRP agonists: A potential mechanism for headache driven by TRPA1 activation

Exposure to airborne toxins can trigger headaches, but the mechanisms are not well understood. Some environmental toxins, such as acrolein, activate transient receptor potential ankyrin 1 (TRPA1), a receptor involved in pain sensation that is highly expressed in the trigeminovascular system. It has been shown in rat models that repeated exposure to acrolein induces trigeminovascular sensitization to both TRPA1 and TRP vanilloid 1 (TRPV1) agonists, a phenomenon linked to headache. In this study, we test the hypothesis that the sensitization of trigeminovascular responses in rats after acrolein exposure via inhalation is associated with changes in levels of endogenous lipids, including TRPV1 agonists, in the trigeminal ganglia, trigeminal nucleus, and cerebellum. Lipidomics analysis of 80 lipids was performed on each tissue after acute acrolein, chronic acrolein, or room air control. Both acute and chronic acrolein exposure drove widespread alterations in lipid levels. After chronic acrolein exposure, levels of all 6N-acyl ethanolamines in the screening library, including the endogenous cannabinoid and TRPV1 agonist, N-arachidonoyl ethanolamine, were elevated in trigeminal tissue and in the cerebellum. This increase in TRPV1 ligands by acrolein exposure may indicate further downstream signaling, in that we also show here that a combination of these TRPV1 endogenous agonists increases the potency of the individual ligands in TRPV1-HEK cells. In addition to these TRPV1 agonists, 3 TRPV3 antagonists, 4 TRPV4 agonists, and 25 orphan lipids were up and down regulated after acrolein exposure. These data support the hypothesis that lipid signaling may represent a mechanism by which repeated exposure to the TRPA1 agonist and environmental toxin, acrolein, drives trigeminovascular sensitization.

ラット脊髄後角膠様質細胞への侵害受容シナプス伝達に対するTRPA1チャネル作動薬の応答解析 ──In vitroおよびin vivoパッチクランプ解析を用いて──

The transient receptor potential ankyrin 1 (TRPA1) channel has been proposed to be a molecular transducer of cold and inflammatory nociceptive signals. It is expressed on a subset of small primary afferent neurons both in the peripheral terminals, where it serves as a sensor, and on the central nerve endings in the dorsal horn. The substantia gelatinosa (SG) of the spinal cord is a key site for integration of noxious inputs. The SG neurons are morphologically and functionally heterogeneous and the precise synaptic circuits of the SG are poorly understood. We examined how activation of TRPA1 channels affects synaptic transmission onto SG neurons using in vitro and in vivo whole–cell patch–clamp recordings in adult rat spinal cord. We have shown that cinnamaldehyde (CA; selective TRPA1 agonist) elicited a barrage of excitatory postsynaptic currents (EPSCs) in a subset of the SG neurons. However, the role of TRPA1 on synaptic transmission in the spinal dorsal horn is not fully understood. In this study, an action of CA was investigated on dorsal root–evoked synaptic transmission to SG neurons in adult rat spinal cord slices by use of the whole–cell voltage–clamp technique. In about 30% of neurons examined, superfusing CA depressed C–fiber–evoked EPSCs. On the contrary, Aδ–fiber–evoked EPSCs were unaffected by CA in all of cells tested. Such a blockade action was not observed when CA was applied in the presence of a specific TRPA1 antagonist, HC–030031. Next, we examined the effects of mechanical stimuli to the skin during TRPA1 activation in the spinal dorsal horn by using in vivo patch clamp technique. Superfusing CA significantly inhibited pinch evoked EPSCs of the skin.

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

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

Oleuropein aglycone enhances UCP1 expression in brown adipose tissue in high-fat-diet-induced obese rats by activating β-adrenergic signaling

Oleuropein is the pungent principle of raw olives. Oleuropein aglycone (OA) is a major phenolic compound in extra virgin olive oil and the absorbed form of oleuropein. We aimed to determine the mechanism underlying the nutritional effects of oleuropein and OA on interscapular brown adipose tissue (IBAT) in rats with high-fat (HF) diet-induced obesity by examining the agonistic activity of oleuropein and OA toward the transient receptor potential ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1). Four-week-old male Sprague–Dawley rats were fed an HF (palm oil 30% wt:wt) diet alone or with oleuropein (HF-O, 1 g/kg diet) for 28 days. In rats fed HF-O compared to HF, urinary noradrenaline, adrenaline and UCP1 levels in IBAT were significantly higher, whereas plasma leptin levels and the total weight of the abdominal cavity adipose tissue were significantly lower. In anaesthetized 7-week-old male Sprague–Dawley rats, the OA (3.8 mg of intravenous injection)-induced increase in plasma noradrenaline secretion was suppressed by TRPA1 or TRPV1 antagonist and by a β2- or β3-adrenoceptor antagonist. Furthermore, OA-activated rat and human TRPV1s expressed on HEK293 cells at the same level as zingerone (pungent component in ginger). OA also activated humanTRPA1, and its potency was approximately 10-fold stronger than that for TRPV1. These findings suggest that OA is the agonist of both TRPA1 and TRPV1 and that OA enhances UCP1 expression in IBAT with a concomitant decrease in the visceral fat mass of HF-diet-induced obese rats through enhanced noradrenaline secretion via β-adrenergic action following TRPA1 and TRPV1 activation.

The transient receptor potential ankyrin-1 mediates mechanical hyperalgesia induced by the activation of B1 receptor in mice

The kinin receptor B1 and the transient receptor potential ankyrin 1 (TRPA1) work as initiators and gatekeepers of nociception and inflammation. This study reports that the nociceptive transmission induced by activation of B1 receptor is dependent on TRPA1 ion channel. The mechanical hyperalgesia was induced by intrathecal (i.t.) injection of B1 agonist des-Arginine9-bradykinin (DABK) or TRPA1 agonist cinnamaldehyde and was evaluated by the withdrawal response after von Frey Hair application in the hind paw. After behavioral experiments, lumbar spinal cord and dorsal root ganglia (DRG) were harvested to assess protein expression and mRNA by immunohistochemistry and real time-PCR, respectively. The pharmacological antagonism (HC030031) or the down-regulation of TRPA1 greatly inhibited the mechanical hyperalgesia induced by DABK. Intrathecal injection of DABK up regulated the ionized calcium binding adaptor molecule (Iba-1) in lumbar spinal cord (L5-L6); TRPA1 protein and mRNA in lumbar spinal cord; and B1 receptor mRNA in both lumbar spinal cord and DRG. The knockdown of TRPA1 prevented microglia activation induced by DABK. Furthermore, the mechanical hyperalgesia induced by either DABK or by cinnamaldehyde was significantly reduced by inhibition of cyclooxygenase (COX), protein kinase C (PKC) or phospholipase C (PLC). In summary, this study revealed that TRPA1 positively modulates the mechanical hyperalgesia induced by B1 receptor activation in the spinal cord and that the classical GPCR downstream molecules PLC, diacylglycerol (DAG), 3,4,5-inositide phosphate (IP3) and PKC are involved in the nociceptive transmission triggered by these two receptors.

Mechanical antihypersensitivity effect induced by repeated spinal administrations of a TRPA1 antagonist or a gap junction decoupler in peripheral neuropathy

Spinal transient receptor potential ankyrin 1 (TRPA1) channel is associated with various pain hypersensitivity conditions. Spinally, TRPA1 is expressed by central terminals of nociceptive nerve fibers and astrocytes. Among potential endogenous agonists of TRPA1 is H2O2 generated by d-amino acid oxidase (DAAO) in astrocytes. Here we studied whether prolonged block of the spinal TRPA1 or astrocytes starting at time of injury attenuates development and/or maintenance of neuropathic hypersensitivity. Additionally, TRPA1 and DAAO mRNA were determined in the dorsal root ganglion (DRG) and spinal dorsal horn (SDH). Experiments were performed in rats with spared nerve injury (SNI) and chronic intrathecal catheter. Drugs were administered twice daily for the first seven injury days or only once seven days after injury. Mechanical hypersensitivity was assessed with monofilaments. Acute and prolonged treatment with Chembridge-5861528 (a TRPA1 antagonist), carbenoxolone (an inhibitor of activated astrocytes), or gabapentin (a comparison drug) attenuated tactile allodynia-like responses evoked by low (2g) stimulus. However, antihypersensitivity effect of these compounds was short of significance at a high (15g) stimulus intensity. No preemptive effects were observed. In healthy controls, carbenoxolone failed to prevent hypersensitivity induced by spinal cinnamaldehyde, a TRPA1 agonist. TRPA1 and DAAO mRNA in the DRG but not SDH were slightly increased in SNI, independent of drug treatment. The results indicate that prolonged peri-injury block of spinal TRPA1 or inhibition of spinal astrocyte activation attenuates maintenance but not development of mechanical (tactile allodynia-like) hypersensitivity after nerve injury.

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

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

Group III mGluR8 negatively modulates TRPA1

Several lines of evidence indicate group III metabotropic glutamate receptors (mGluRs) have systemic anti-hyperalgesic effects. We hypothesized this could occur through modulation of TRP channels on nociceptors. This study used a multifaceted approach to examine the interaction between group III mGluRs (mGluR8) and transient receptor potential ankyrin 1 (TRPA1) on cutaneous nociceptors in rats. Ca2+ imaging studies demonstrated co-localization and functional coupling of TRPA1 and mGluR8, since 1μM (S)-3,4-dicarboxyphenylglycine (DCPG) (mGluR8 agonist) significantly reduced Ca2+ mobilization produced by 30μM mustard oil (MO), a TRPA1 agonist. Behavioral studies demonstrated that 10mM MO produced mechanical hypersensitivity when topically applied to the hind paw, significantly decreasing paw withdrawal threshold (PWT) from 15g to 6g. However, administration of 30μM DCPG prior to 10mM MO reversed this hypersensitivity such that PWT was not significantly different from baseline. At the single-fiber level, compared to vehicle, 30μM MO significantly increased nociceptor activity and decreased mechanical threshold. However, 30μM DCPG reversed both of these MO-induced effects. Furthermore, DCPG significantly reduced the number of MO-induced mechanically sensitive fibers. Inhibition of protein kinase A (PKA) using Rp-cyclic 3′,5′-hydrogen phosphorothioate adenosine triethylammonium salt (RpCAMPS) (PKA inhibitor, 1 and 10μM) significantly reduced MO-induced Ca2+ mobilization. Taken together, these results show that group III mGluRs negatively modulate TRPA1 activity on cutaneous nociceptors. Furthermore, it is likely that this modulation occurs intracellularly at the level of the cAMP/PKA pathway. This study demonstrates that group III agonists may be effective in the treatment of mechanical hypersensitivity which can develop as a result of inflammation, nerve injury, chemotherapy and other disease states.

Carbonyl stress-induced 5-hydroxytriptamine secretion from RIN-14B, rat pancreatic islet tumor cells, via the activation of transient receptor potential ankyrin 1.

Methylglyoxal (MG), a highly reactive dicarbonyl substance, is known as an endogenous carbonyl stress-inducing substance related to various disease states. Irritable bowel syndrome (IBS) is one of the most frequently encountered gastrointestinal disorders and MG is considered to be its causal substance. An increased serum 5-hydroxytryptamine (5-HT) level is related to IBS symptoms and the majority of 5-HT originates from enterochromaffin (EC) cells in the intestine. Here we examine the mechanisms of MG-induced 5-HT secretion using RIN-14B cells derived from a rat pancreatic islet tumor since these cells are used as a model for EC cells. MG increased the intracellular Ca(2+) concentration ([Ca(2+)]i) and 5-HT secretion, both of which were inhibited by the removal of extracellular Ca(2+) and specific transient receptor potential ankyrin 1 (TRPA1) antagonists. MG elicited an inward current under voltage-clamped conditions. Prior application of MG evoked reciprocal suppression of subsequent [Ca(2+)]i responses to allylisothiocyanate, a TRPA1 agonist, and vice versa. Glyoxal, an analog of MG, also evoked [Ca(2+)]i and secretory responses but its potency was much lower than that of MG. The present results suggest that MG promotes 5-HT secretion through the activation of TRPA1 in RIN-14B cells. These results may indicate that TRPA1 is a promising target for the treatment of IBS and that the RIN-14B cell line is a useful model for investigation of IBS.

Diet-induced obesity alters dural CGRP release and potentiates TRPA1-mediated trigeminovascular responses.

Background Clinical studies suggest a link between obesity and the primary headache disorder migraine. In our study we aimed to reveal the effect of obesity on meningeal nociceptor function in rats receiving a high-fat, high-sucrose diet. Methods Transient receptor potential ankyrin 1 (TRPA1) receptor activation-induced changes in meningeal blood flow, release of calcitonin gene-related peptide (CGRP) from trigeminal afferents and TRPA1 protein expression in the trigeminal ganglia were measured in control and obese rats. Metabolic parameters of the animals were assessed by measuring glucose and insulin homeostasis as well as plasma cytokine concentrations. Results The present experiments revealed an enhanced basal and TRPA1 receptor agonist-induced CGRP release from meningeal afferents of obese insulin-resistant rats and an attenuated CGRP release to potassium chloride. Obesity was also associated with an augmented vasodilatation in meningeal arteries after dural application of the TRPA1 agonist acrolein, a reduction in TRPA1 protein expression in the trigeminal ganglia and elevations in circulating proinflammatory cytokines IL-1β and IL-6 in addition to increased fasting blood glucose and insulin concentrations. Conclusions Our results suggest trigeminal sensitisation as a mechanism for enhanced headache susceptibility in obese individuals after chemical exposure of trigeminal nociceptors.