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Background: Diabetic cardiac autonomic neuropathy (DCAN) is a serious diabetic complication with no approved pharmacological agents for its treatment. Parasympathetic system dysfunction characterized by vagal nerve damage is one of the major drivers of DCAN. The TRPC5 or transient receptor potential canonical 5 channel is a promising target in autonomic dysfunction; however, its role in vagal nerve damage and subsequent DCAN has not yet been elucidated. The present study investigated the role of the TRPC5 channel in DCAN using [N-{3-(adamantan-2-yloxy)-propyl}-3-(6-methyl-1,1-dioxo-2H-1λ 6 ,2,4-benzothiadiazin-3-yl) propanamide)] or BTD, which is a potent TRPC5 activator., Objectives: The role of the TRPC5 channel and its activator, BTD, was investigated in the treatment of parasympathetic dysfunction associated with DCAN., Methods: Type 1 diabetes was induced in male Sprague-Dawley rats using streptozotocin. The alterations in cardiac autonomic parameters in diabetic animals were assessed by heart rate variability, hemodynamic parameters, and baroreflex sensitivity. TRPC5's role in DCAN was investigated by treating diseased rats with BTD (1 and 3 mg/kg, i.p. for 14 days). BTD's beneficial effects in parasympathetic dysfunction were assessed by western blotting, estimating oxidative stress and inflammatory markers in the vagus nerve., Results: BTD treatment (3 mg/kg, i.p.) once daily for 14 days ameliorated heart rate variability, hemodynamic dysfunction, and baroreflex sensitivity in diseased rats. BTD treatment down regulated TRPC5 expression by increasing the activity of protein kinase C in the vagus nerve. It also down-regulated the apoptotic marker CASPASE-3 and also exerted potent anti-inflammatory action on pro-inflammatory cytokines levels in the vagus., Conclusion: BTD ameliorated parasympathetic dysfunction associated with DCAN by virtue of its TRPC5 modulatory, anti-inflammatory, and anti-apoptotic properties., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Increased blood pressure variability (BPV) has been proved to be associated with cardiovascular morbidity and mortality. It is of great significance to elucidate the mechanism of BPV increase. The cation channel transient receptor potential canonical 6 (TRPC6) is involved in a series of cardiovascular disease. Our experiment aimed to explore the role of TRPC6 in the development of BPV increase. Sino-aortic denervation (SAD) operation was applied to establish the model of BPV increase in rats. The BPV was presented as the standard deviation to the mean of systolic or diastolic blood pressure every 1 h during 12 h of the light period. SAD was performed in male Sprague Dawley (SD) rats at the age of 10 weeks. At 8 weeks after SAD operation, the hemodynamic parameters were determined non-invasively via a Rodent Blood Pressure Analysis System. The TRPC6 expressions in myocardial and thoracic aortic tissue was determined utilizing Western Blot, immunofluorescence and quantitative RT-PCR. The expression of TRPC3 was detected as well. To investigate whether TRPC6 was a causative factor of BPV increase in SAD rats, TRPC6 activator and inhibitor with three progressively increasing doses were intraperitoneally injected to the SAD rats. We found that SAD rats presented significant augmentation of systolic and diastolic BPV with no change of BP level and heart rate. The mRNA and protein expression levels of TRPC6 in myocardial and thoracic aortic tissue in SAD rats were substantially increased, but there was no obvious change in TRPC3 expression. The systolic and diastolic BPV increase were dose-dependently exacerbated after TRPC6 activation with GSK1702934A but were dose-dependently attenuated after TRPC6 inhibition with SAR7334. In Conclusion, the TRPC6 (but not TRPC3) expressions in myocardial and thoracic aortic tissue were substantially increased in SAD rats, and TRPC6 probably played an important role in the development of BPV elevation.

Canonical or classical transient receptor potential 4 and 5 proteins (TRPC4 and TRPC5) assemble as homomers or heteromerize with TRPC1 protein to form functional nonselective cationic channels with high calcium permeability. These channel complexes, TRPC1/4/5, are widely expressed in nervous and cardiovascular systems, also in other human tissues and cell types. It is debatable that TRPC1 protein is able to form a functional ion channel on its own. A recent explosion of molecular information about TRPC1/4/5 has emerged including knowledge of their distribution, function, and regulation suggesting these three members of the TRPC subfamily of TRP channels play crucial roles in human physiology and pathology. Therefore, these ion channels represent potential drug targets for cancer, epilepsy, anxiety, pain, and cardiac remodelling. In recent years, a number of highly selective small-molecule modulators of TRPC1/4/5 channels have been identified as being potent with improved pharmacological properties. This review will focus on recent remarkable small-molecule agonists: (-)-englerin A and tonantzitlolone and antagonists: Pico145 and HC7090, of TPRC1/4/5 channels. In addition, this work highlights other recently identified modulators of these channels such as the benzothiadiazine derivative, riluzole, ML204, clemizole, and AC1903. Together, these treasure troves of agonists and antagonists of TRPC1/4/5 channels provide valuable hints to comprehend the functional importance of these ion channels in native cells and in vivo animal models. Importantly, human diseases and disorders mediated by these proteins can be studied using these compounds to perhaps initiate drug discovery efforts to develop novel therapeutic agents., (© 2019 The British Pharmacological Society.)

Background and Purpose: The diterpene ester tonantzitlolone (TZL) is a natural product, which displays cytotoxicity towards certain types of cancer cell such as renal cell carcinoma cells. The effect is similar to that of (-)-englerin A, and so, although it is chemically distinct, we investigated whether TZL also targets transient receptor potential canonical (TRPC) channels of the 1, 4 and 5 type (TRPC1/4/5 channels)., Experimental Approach: The effects of TZL on renal cell carcinoma A498 cells natively expressing TRPC1 and TRPC4, modified HEK293 cells overexpressing TRPC4, TRPC5, TRPC4-TRPC1 or TRPC5-TRPC1 concatemer, TRPC3 or TRPM2, or CHO cells overexpressing TRPV4 were studied by determining changes in intracellular Ca 2+ , or whole-cell or excised membrane patch-clamp electrophysiology., Key Results: TZL induced an elevation of intracellular Ca 2+ in A498 cells, similar to that evoked by englerin A. TZL activated overexpressed channels with EC 50 values of 123 nM (TRPC4), 83 nM (TRPC5), 140 nM (TRPC4-TRPC1) and 61 nM (TRPC5-TRPC1). These effects of TZL were reversible on wash-out and potently inhibited by the TRPC1/4/5 inhibitor Pico145. TZL activated TRPC5 channels when bath-applied to excised outside-out but not inside-out patches. TZL failed to activate endogenous store-operated Ca 2+ entry or overexpressed TRPC3, TRPV4 or TRPM2 channels in HEK 293 cells., Conclusions and Implications: TZL is a novel potent agonist for TRPC1/4/5 channels, which should be useful for testing the functionality of this type of ion channel and understanding how TRPC1/4/5 agonists achieve selective cytotoxicity against certain types of cancer cell., (© 2018 The British Pharmacological Society.)

Transient receptor potential channel 5 (TRPC5) is highly expressed in brain and kidney and mediates calcium influx and promotes cell migration. In the kidney, loss of TRPC5 function has been reported to benefit kidney filter dynamics by balancing podocyte cytoskeletal remodeling. However, in vivo gain-in-function studies of TRPC5 with respect to kidney function have not been reported. To address this gap, we developed two transgenic mouse models on the C57BL/6 background by overexpressing either wild-type TRPC5 or a TRPC5 ion-pore mutant. Compared with nontransgenic controls, neither transgenic model exhibited an increase in proteinuria at 8 months of age or a difference in LPS-induced albuminuria. Moreover, activation of TRPC5 by Englerin A did not stimulate proteinuria, and inhibition of TRPC5 by ML204 did not significantly lower the level of LPS-induced proteinuria in any group. Collectively, these data suggest that the overexpression or activation of the TRPC5 ion channel does not cause kidney barrier injury or aggravate such injury under pathologic conditions., (Copyright © 2018 by the American Society of Nephrology.)

The transient receptor potential canonical channel 5 (TRPC5) is a Ca 2+ -permeable ion channel, which is predominantly expressed in the brain. TRPC5-deficient mice exhibit a reduced innate fear response and impaired motor control. In addition, outgrowth of hippocampal and cerebellar neurons is retarded by TRPC5. However, pharmacological evidence of TRPC5 function on cellular or organismic levels is sparse. Thus, there is still a need for identifying novel and efficient TRPC5 channel modulators. We, therefore, screened compound libraries and identified the glucocorticoid methylprednisolone and N-[3-(adamantan-2-yloxy)propyl]-3-(6-methyl-1,1-dioxo-2H-1λ 6 ,2,4-benzothiadiazin-3-yl)propanamide (BTD) as novel TRPC5 activators. Comparisons with closely related chemical structures from the same libraries indicate important substructures for compound efficacy. Methylprednisolone activates TRPC5 heterologously expressed in HEK293 cells with an EC 50 of 12μM, while BTD-induced half-maximal activation is achieved with 5-fold lower concentrations, both in Ca 2+ assays (EC 50 =1.4μM) and in electrophysiological whole cell patch clamp recordings (EC 50 =1.3 μM). The activation resulting from both compounds is long lasting, reversible and sensitive to clemizole, a recently established TRPC5 inhibitor. No influence of BTD on homotetrameric members of the remaining TRPC family was observed. On the main sensory TRP channels (TRPA1, TRPV1, TRPM3, TRPM8) BTD exerts only minor activity. Furthermore, BTD can activate heteromeric channel complexes consisting of TRPC5 and its closest relatives TRPC1 or TRPC4, suggesting a high selectivity of BTD for channel complexes bearing at least one TRPC5 subunit., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The nonselective cation channel transient receptor potential ankryn subtype family 1 (TRPA1) is expressed in neurons of dorsal root ganglia and trigeminal ganglia and also in vagal afferent neurons that innervate the lungs and gastrointestinal tract. Many TRPA1 agonists are reactive electrophilic compounds that form covalent adducts with TRPA1. Allyl isothiocyanate (AITC), the common agonist used to identify TRPA1, contains an electrophilic group that covalently binds with cysteine residues of TRPA1 and confers a structural change on the channel. There is scientific motivation to identify additional compounds that can activate TRPA1 with different mechanisms of channel gating. We provide evidence that ethyl vanillin (EVA) is a TRPA1 agonist. Using fluorescent calcium imaging and whole-cell patch-clamp electrophysiology on dissociated rat vagal afferent neurons and TRPA1-transfected COS-7 cells, we discovered that EVA activates cells also activated by AITC. Both agonists display similar current profiles and conductances. Pretreatment with A967079, a selective TRPA1 antagonist, blocks the EVA response as well as the AITC response. Furthermore, EVA does not activate vagal afferent neurons from TRPA1 knockout mice, showing selectivity for TRPA1 in this tissue. Interestingly, EVA appears to be pharmacologically different from AITC as a TRPA1 agonist. When AITC is applied before EVA, the EVA response is occluded. However, they both require intracellular oxidation to activate TRPA1. These findings suggest that EVA activates TRPA1 but via a distinct mechanism that may provide greater ease for study in native systems compared with AITC and may shed light on differential modes of TRPA1 gating by ligand types., (Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.)

Transient receptor potential canonical 3/6/7 (TRPC3/6/7) are highly homologous receptor-operated nonselective cation channels. Despite their physiological significance, very few selective and potent agonists are available for functional examination of these channels. Using a cell-based high throughput screening approach, a lead compound with the pyrazolopyrimidine skeleton was identified as a TRPC6 agonist. Synthetic schemes for the lead and its analogues were established, and structural-activity relationship studies were carried out. A series of potent and direct agonists of TRPC3/6/7 channels were identified, and among them, 4m-4p have a potency order of TRPC3 > C7 > C6, with 4n being the most potent with an EC 50 of <20 nM on TRPC3. Importantly, these compounds exhibited no stimulatory activity on related TRP channels. The potent and selective compounds described here should be suitable for evaluation of the roles of TRPC channels in the physiology and pathogenesis of diseases, including glomerulosclerosis and cancer.

Background: Although TRPA1, SP, histamine and 5-hydroxytryptamine (5-HT) have recognized contribution to nociceptive mechanisms, little is known about how they interact with each other to mediate inflammatory pain in vivo. In this study we evaluated whether TRPA1, SP, histamine and 5-HT interact, in an interdependent way, to induce nociception in vivo., Methods and Results: The subcutaneous injection of the TRPA1 agonist allyl isothiocyanate (AITC) into the rat's hind paw induced a dose-dependent and short lasting behavioral nociceptive response that was blocked by the co-administration of the TRPA1 antagonist, HC030031, or by the pretreatment with antisense ODN against TRPA1. AITC-induced nociception was significantly decreased by the co-administration of selective antagonists for the NK1 receptor for substance P, the H1 receptor for histamine and the 5-HT 1A or 3 receptors for 5-HT. Histamine- or 5-HT-induced nociception was decreased by the pretreatment with antisense ODN against TRPA1. These findings suggest that AITC-induced nociception depends on substance P, histamine and 5-HT, while histamine- or 5-HT-induced nociception depends on TRPA1. Most important, AITC interact in a synergistic way with histamine, 5-HT or substance P, since their combination at non-nociceptive doses induced a nociceptive response much higher than that expected by the sum of the effect of each one alone. This synergistic effect is dependent on the H1, 5-HT 1A or 3 receptors., Conclusion: Together, these findings suggest a self-sustainable cycle around TRPA1, no matter where the cycle is initiated each step is achieved and even subeffective activation of more than one step results in a synergistic activation of the overall cycle.

We previously showed that magnesium sulfate (MS) has systemic antinociceptive and local peripheral pronociceptive effects. The role of transient receptor potential (TRP) channels and acid-sensing ion channels (ASICs) in the mechanism of action of MS has not been investigated in detail. The aim of this study was to explore the participation of TRP channels in the pronociceptive action of MS in rats after its intraplantar injection. The paw withdrawal threshold (PWT) to mechanical stimuli was measured by the electronic von Frey test. Drugs that were tested were either co-administered with an isotonic pH-unadjusted or pH-adjusted solution of MS intraplantarily, or to the contralateral paw to exclude systemic effects. We found that the subcutaneous administration of both pH-adjusted (7.4) and pH-unadjusted (about 6.0) isotonic (6.2% w/v in water) solutions of MS induce the pain at the injection site. The pH-unadjusted MS solution-induced mechanical hyperalgesia decreased in a dose-dependent manner as a consequence of co-injection of capsazepine, a selective TRPV1 antagonist (20, 100 and 500pmol/paw), RN-1734, a selective TRPV4 antagonist (1.55, 3.1 and 6.2μmol/paw), HC-030031, a selective TRPA1 antagonist (5.6, 28.1 and 140nmol/paw), and amiloride hydrochloride, a non-selective ASIC inhibitor (0.83, 2.5 and 7.55μmol/paw). In pH-adjusted MS-induced hyperalgesia, the highest doses of TRPV1, TRPV4 and TRPA1 antagonists displayed effects that were, respectively, either similar, less pronounced or delayed in comparison to the effect induced by administration of the pH-unadjusted MS solution; the ASIC antagonist did not have any effect. These results suggest that the MS-induced local peripheral mechanical hyperalgesia is mediated via modulation of the activity of peripheral TRPV1, TRPV4, TRPA1 and ASICs. Specific local inhibition of TRP channels represents a novel approach to treating local injection-related pain., (Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.)

Mushroom dendritic spine structures are essential for memory storage and the loss of mushroom spines may explain memory defects in aging and Alzheimer's disease (AD). The stability of mushroom spines depends on stromal interaction molecule 2 (STIM2)-mediated neuronal-store-operated Ca 2+ influx (nSOC) pathway, which is compromised in AD mouse models, in aging neurons, and in sporadic AD patients. Here, we demonstrate that the Transient Receptor Potential Canonical 6 (TRPC6) and Orai2 channels form a STIM2-regulated nSOC Ca 2+ channel complex in hippocampal mushroom spines. We further demonstrate that a known TRPC6 activator, hyperforin, and a novel nSOC positive modulator, NSN21778 (NSN), can stimulate activity of nSOC pathway in the spines and rescue mushroom spine loss in both presenilin and APP knock-in mouse models of AD. We further show that NSN rescues hippocampal long-term potentiation impairment in APP knock-in mouse model. We conclude that the STIM2-regulated TRPC6/Orai2 nSOC channel complex in dendritic mushroom spines is a new therapeutic target for the treatment of memory loss in aging and AD and that NSN is a potential candidate molecule for therapeutic intervention in brain aging and AD., Significance Statement: Mushroom dendritic spine structures are essential for memory storage and the loss of mushroom spines may explain memory defects in Alzheimer's disease (AD). This study demonstrated that Transient Receptor Potential Canonical 6 (TRPC6) and Orai2 form stromal interaction molecule 2 (STIM2)-regulated neuronal-store-operated Ca 2+ influx (nSOC) channel complex in hippocampal synapse and the resulting Ca 2+ influx is critical for long-term maintenance of mushroom spines in hippocampal neurons. A novel nSOC-positive modulator, NSN21778 (NSN), rescues mushroom spine loss and synaptic plasticity impairment in AD mice models. The TRPC6/Orai2 nSOC channel complex is a new therapeutic target and NSN is a potential candidate molecule for therapeutic intervention in brain aging and AD., (Copyright © 2016 the authors 0270-6474/16/3611837-14$15.00/0.)

Besides their deleterious action on cardiac muscle, anthracycline-type cytostatic agents exert significant neurotoxic effects on primary sensory neurons. Since cardiac sensory nerves confer protective effects on heart muscle and share common traits with cutaneous chemosensitive nerves, this study examined the effects of cardiotoxic doses of adriamycin on the function and morphology of epidermal nerves. Sensory neurogenic vasodilatation, plasma extravasation, and the neural CGRP release evoked by TRPV1 and TRPA1 agonists in vitro were examined by using laser Doppler flowmetry, the Evans blue technique, and ELISA, respectively. Carrageenan-induced hyperalgesia was assessed with the Hargreaves method. Immunohistochemistry was utilized to study cutaneous innervation. Adriamycin treatment resulted in profound reductions in the cutaneous neurogenic sensory vasodilatation and plasma extravasation evoked by the TRPV1 and TRPA1 agonists capsaicin and mustard oil, respectively. The in vitro capsaicin-, but not high potassium-evoked neural release of the major sensory neuropeptide, CGRP, was markedly attenuated after adriamycin treatment. Carrageenan-induced inflammatory hyperalgesia was largely abolished following the administration of adriamycin. Immunohistochemistry revealed a substantial loss of epidermal TRPV1-expressing nociceptive nerves and a marked thinning of the epidermis. These findings indicate impairments in the functions of TRPV1 and TRPA1 receptors expressed on cutaneous chemosensitive nociceptive nerves and the loss of epidermal axons following the administration of cardiotoxic doses of adriamycin. Monitoring of the cutaneous nociceptor function in the course of adriamycin therapy may well be of predictive value for early detection of the deterioration of cardiac nerves which confer protection against the deleterious effects of the drug.

Methylglyoxal (MG) is a reactive carbonyl compound generated in diabetes mellitus. MG is an established transient receptor potential ankyrin 1 (TRPA1) channel agonist that contributes to TRPA1-mediated diabetic pain hypersensitivity. Here we studied whether exposure to diabetes and thereby to elevated endogenous MG modulates hypersensitivity induced by intradermal MG. Moreover, since diabetes induces endoplasmic reticulum (ER) stress, we compared the role of TRPA1 in diabetes and ER stress by assessing whether tunicamycin-induced ER stress, without diabetes, produces TRPA1-mediated pain hypersensitivity and by assessing whether ER stress and diabetes have similar modulatory effects on MG-induced hypersensitivity. In vitro patch clamp recording was performed to assess whether tunicamycin is a TRPA1 agonist. Behavioral tests showed that mechanical hypersensitivity induced by MG is reduced in diabetes and ER stress. In healthy controls, hypersensitivity induced by MG was reduced when MG was administered for the second time in the same but not adjacent plantar sites. Hypersensitivity induced by ER stress was reversed by pharmacological blocking of TRPA1. In vitro patch clamp recording indicated that tunicamycin itself (30 μM) is not a TRPA1 agonist. The results indicate that pain hypersensitivity induced by non-diabetic ER stress as well as that induced by diabetes is mediated TRPA1. Reduction of MG-induced hypersensitivity in diabetes or ER stress may, at least partly, be explained by peripheral mechanisms.

Background: Administration of onabotulinumtoxinA (BoNT-A) to peripheral tissues outside the calvaria reduces the number of days chronic migraine patients experience headache. Because the headache phase of a migraine attack, especially those preceded by aura, is thought to involve activation of meningeal nociceptors by endogenous stimuli such as changes in intracranial pressure (i.e. mechanical) or chemical irritants that appear in the meninges as a result of a yet-to-be-discovered sequence of molecular/cellular events triggered by the aura, we sought to determine whether extracranial injections of BoNT-A alter the chemosensitivity of meningeal nociceptors to stimulation of their intracranial receptive fields., Material and Methods: Using electrophysiological techniques, we identified 161 C- and 135 Aδ-meningeal nociceptors in rats and determined their mechanical response threshold and responsiveness to chemical stimulation of their dural receptive fields with TRPV1 and TRPA1 agonists seven days after BoNT-A administration to different extracranial sites. Two paradigms were compared: distribution of 5 U BoNT-A to the lambdoid and sagittal sutures alone, and 1.25 U to the sutures and 3.75 U to the temporalis and trapezius muscles., Results: Seven days after it was administered to tissues outside the calvaria, BoNT-A inhibited responses of C-type meningeal nociceptors to stimulation of their intracranial dural receptive fields with the TRPV1 agonist capsaicin and the TRPA1 agonist mustard oil. BoNT-A inhibition of responses to capsaicin was more effective when the entire dose was injected along the suture lines than when it was injected into muscles and sutures. As in our previous study, BoNT-A had no effect on non-noxious mechanosensitivity of C-fibers or on responsiveness of Aδ-fibers to mechanical and chemical stimulation., Discussion: This study demonstrates that extracranial administration of BoNT-A suppresses meningeal nociceptors' responses to stimulation of their intracranial dural receptive fields with capsaicin and mustard oil. The findings suggest that surface expression of TRPV1 and TRPA1 channels in dural nerve endings of meningeal nociceptors is reduced seven days after extracranial administration of BoNT-A. In the context of chronic migraine, reduced sensitivity to molecules that activate meningeal nociceptors through the TRPV1 and TRPA1 channels can be important for BoNT-A's ability to act as a prophylactic., (© International Headache Society 2016.)

The primary complaint of burn victims is an intense, often devastating spontaneous pain, with persistence of mechanical and thermal allodynia. The transient receptor potential channels, TRPV1 and TRPA1, are expressed by a subset of nociceptive sensory neurons and contribute to inflammatory hypersensitivity. Although their function in the periphery is well known, a role for these TRP channels in central pain mechanisms is less well defined. Lipid agonists of TRPV1 are released from peripheral tissues via enzymatic oxidation after burn injury; however, it is not known if burn injury triggers the release of oxidized lipids in the spinal cord. Accordingly, we evaluated whether burn injury evoked the central release of oxidized lipids . Analysis of lipid extracts of spinal cord tissue with HPLC-MS revealed a significant increase in levels of the epoxide and diol metabolites of linoleic acid: 9,10-DiHOME, 12,13-DiHOME, 9(10)-EpOME, and 12(13)-EpOME, that was reduced after intrathecal (i.t.) injection of the oxidative enzyme inhibitor ketoconazole. Moreover, we found that these four lipid metabolites were capable of specifically activating both TRPV1 and TRPA1. Intrathecal injection of specific antagonists to TRPV1 (AMG-517) or TRPA1 (HC-030031) significantly reduced post-burn mechanical and thermal allodynia. Finally, i.t. injection of ketoconazole significantly reversed post-burn mechanical and thermal allodynia. Our data indicate that spinal cord TRPV1 and TRPA1 contributes to pain after burn and identifies a novel class of oxidized lipids elevated in the spinal cord after burn injury. Since the management of burn pain is problematic, these findings point to a novel approach for treating post-burn pain.

Anoctamin-1 [ANO1, also known as transmembrane protein 16A (TMEM16A)] is a Ca(2+)-activated Cl(-) channel expressed in arterial myocytes that regulates membrane potential and contractility. Signaling mechanisms that control ANO1 activity in arterial myocytes are poorly understood. In cerebral artery myocytes, ANO1 channels are activated by local Ca(2+) signals generated by plasma membrane nonselective cation channels, but the molecular identity of these proteins is unclear. Arterial myocytes express several different nonselective cation channels, including multiple members of the transient receptor potential receptor (TRP) family. The goal of this study was to identify localized ion channels that control ANO1 currents in cerebral artery myocytes. Coimmunoprecipitation and immunofluorescence resonance energy transfer microscopy experiments indicate that ANO1 and canonical TRP 6 (TRPC6) channels are present in the same macromolecular complex and localize in close spatial proximity in the myocyte plasma membrane. In contrast, ANO1 is not near TRPC3, TRP melastatin 4, or inositol trisphosphate receptor 1 channels. Hyp9, a selective TRPC6 channel activator, stimulated Cl(-) currents in myocytes that were blocked by T16Ainh-A01, an ANO1 inhibitor, ANO1 knockdown using siRNA, and equimolar replacement of intracellular EGTA with BAPTA, a fast Ca(2+) chelator that abolishes local Ca(2+) signaling. Hyp9 constricted pressurized cerebral arteries, and this response was attenuated by T16Ainh-A01. In contrast, T16Ainh-A01 did not alter depolarization-induced (60 mM K(+)) vasoconstriction. These data indicate that TRPC6 channels generate a local intracellular Ca(2+) signal that activates nearby ANO1 channels in myocytes to stimulate vasoconstriction., (Copyright © 2016 the American Physiological Society.)

Transient receptor potential canonical (TRPC) proteins form Ca(2+)-permeable cation channels activated upon stimulation of metabotropic receptors coupled to phospholipase C. Among the TRPC subfamily, TRPC3 and TRPC6 channels activated directly by diacylglycerol (DAG) play important roles in brain-derived neurotrophic factor (BDNF) signaling, promoting neuronal development and survival. In various disease models, BDNF restores neurologic deficits, but its therapeutic potential is limited by its poor pharmacokinetic profile. Elucidation of a framework for designing small molecules, which elicit BDNF-like activity via TRPC3 and TRPC6, establishes a solid basis to overcome this limitation. We discovered, through library screening, a group of piperazine-derived compounds that activate DAG-activated TRPC3/TRPC6/TRPC7 channels. The compounds [4-(5-chloro-2-methylphenyl)piperazin-1-yl](3-fluorophenyl)methanone (PPZ1) and 2-[4-(2,3-dimethylphenyl)piperazin-1-yl]-N-(2-ethoxyphenyl)acetamide (PPZ2) activated, in a dose-dependent manner, recombinant TRPC3/TRPC6/TRPC7 channels, but not other TRPCs, in human embryonic kidney cells. PPZ2 activated native TRPC6-like channels in smooth muscle cells isolated from rabbit portal vein. Also, PPZ2 evoked cation currents and Ca(2+) influx in rat cultured central neurons. Strikingly, both compounds induced BDNF-like neurite growth and neuroprotection, which were abolished by a knockdown or inhibition of TRPC3/TRPC6/TRPC7 in cultured neurons. Inhibitors of Ca(2+) signaling pathways, except calcineurin, impaired neurite outgrowth promotion induced by PPZ compounds. PPZ2 increased activation of the Ca(2+)-dependent transcription factor, cAMP response element-binding protein. These findings suggest that Ca(2+) signaling mediated by activation of DAG-activated TRPC channels underlies neurotrophic effects of PPZ compounds. Thus, piperazine-derived activators of DAG-activated TRPC channels provide important insights for future development of a new class of synthetic neurotrophic drugs., (Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.)

A sensitive response of the nervous system to changes in temperature is of predominant importance for homeotherms to maintain a stable body temperature. A number of temperature-sensitive transient receptor potential (TRP) ion channels have been studied as nociceptors that respond to extreme temperatures and harmful chemicals. Recent findings in the field of pain have established a family of six thermo-TRP channels (TRPA1, TRPM8, TRPV1, TRPV2, TRPV3, and TRPV4) that exhibit sensitivity to increases or decreases in temperature, as well as to chemical substances eliciting the respective hot or cold sensations. In this study, we used behavioral methods to investigate whether mustard oil (allyl isothiocyanate) and capsaicin affect the sensitivity to heat, innocuous and noxious cold, and mechanical stimuli in male rats. The results obtained indicate that TRPA1 and TRPV1 channels are clearly involved in pain reactions, and the TRPA1 agonist allyl isothiocyanate enhances the heat pain sensitivity, possibly by indirectly modulating TRPV1 channels coexpressed in nociceptors with TRPA1. Overall, our data support the role of thermosensitive TRPA1 and TRPV1 channels in pain modulation and show that these two thermoreceptor channels are in a synergistic and/or conditional relationship with noxious heat and cold cutaneous stimulation.

Injury to podocytes is considered a major contributor to diabetic kidney disease: their loss causes proteinuria and progressive glomerulosclerosis. Podocyte depletion may result from improper calcium handling due to abnormal activation of the calcium permeant TRPC (Transient Receptor Potential Canonical) channels. Angiotensin II (Ang II) levels are found to be elevated in diabetes; furthermore, it was reported that Ang II causes activation of TRPC6 in podocytes. We hypothesized here that Ang II-mediated calcium influx is aggravated in the podocytes under the conditions of type 1 diabetic nephropathy (DN). Diabetes was induced in the Dahl Salt-Sensitive rats by an injection of streptozotocin (STZ-SS). Eleven weeks post treatment was sufficient for the animals to develop hyperglycemia, excessive urination, weight loss, microalbuminuria, nephrinuria and display renal histological lesions typical for patients with DN. Patch-clamp electrophysiology performed on podocytes of the freshly isolated glomeruli showed enhanced basal TRPC channel activity in the STZ-SS rats, and increased response to Ang II; total calcium influx triggered by Ang II application was also augmented in podocytes of these rats. Our studies have a strong potential for advancing the understanding of TRPC-mediated effects on podocytopenia in DN initiation.

During clinical development of analgesics, it is important to have access to pharmacologically specific human pain models. o-Chlorobenzylidene malononitrile (CS) is a selective and potent agonist of the transient receptor potential ankyrin repeat 1 (TRPA1), which is a transducer molecule in nociceptors sensing reactive chemical species. While CS has been subject to extensive toxicological investigations in animals and human beings, its effects on intradermal or subcutaneous injection have not previously been reported. We have investigated the potential of CS to be used as an agonist on TRPA1 in human experimental pain studies. A calcium influx assay was used to confirm the capacity of CS to activate TRPA1 with >100,000 times the selectivity over the transient receptor potential vanilloid receptor 1. CS dose-dependently (EC50 0.9 μM) released calcitonin gene-related peptide in rat dorsal root ganglion cultures, supporting involvement in pain signalling. In a local tolerance study, injection of a single intradermal dose of 20 mM CS to rats resulted in superficial, circular crusts at the injection sites after approximately 4 days. The histopathology evaluation revealed a mild, acute inflammatory reaction in the epidermis and dermis at the intradermal CS injection site 1 day after administration. After 14 days, the epidermal epithelium was fully restored. The symptoms were not considered to be adverse, and it is suggested that doses up to 20 μL of 20 mM CS can be safely administered to human beings. In conclusion, our data support development of a CS human dermal pain model., (© 2015 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).)

Citronellal, a well-known plant-derived mosquito repellent, was previously reported to repel Drosophila melanogaster via olfactory pathways involving but not directly activating Transient Receptor Potential Ankyrin 1 (TRPA1). Here, we show that citronellal is a direct agonist for Drosophila and human TRPA1s (dTRPA1 and hTRPA1) as well as Anopheles gambiae TRPA1 (agTRPA1). Citronellal-induced activity is isoform-dependent for Drosophila and Anopheles gambiae TRPA1s. The recently identified dTRPA1(A) and ag-TRPA1(A) isoforms showed citronellal-provoked currents with EC50s of 1.0 B1 0.2 and 0.1 B1 0.03 mM, respectively, in Xenopus oocytes, while the sensitivities of TRPA1(B)s were much inferior to those of TRPA1(A)s. Citronellal dramatically enhanced the feeding-inhibitory effect of the TRPA1 agonist N-methylmaleimide (NMM) in Drosophila at an NMM concentration that barely repels flies. Thus, citronellal can promote feeding deterrence of fruit flies through direct action on gustatory dTRPA1, revealing the first isoform-specific function for TRPA1(A).

The transient receptor potential (TRP) channels are widely distributed in the central nervous system (CNS) and peripheral nervous system. We examined the effects of TRP ankyrin 1 (TRPA1) agonists (cinnamaldehyde and allyl isothiocyanate) on respiratory rhythm generation in brainstem-spinal cord preparations from newborn rats [postnatal days 0-3 (P0-P3)] and in in situ-perfused preparations from juvenile rats (P11-P13). Preparations were superfused with modified Krebs solution at 25-26°C, and activity of inspiratory C4 ventral root (or phrenic nerve) was monitored. In the newborn rat, an in vitro preparation of cinnamaldehyde (0.5 mM) induced typically biphasic responses in C4 rate: an initial short increase and subsequent decrease, then a gradual recovery of rhythm during 15 min of bath application. After washout, the respiratory rhythm rate further increased, remaining 200% of control for >120 min, indicating long-lasting facilitation. Allyl isothiocyanate induced effects similar to those of cinnamaldehyde. The long-lasting facilitation of respiratory rhythm was partially antagonized by the TRPA1 antagonist HC-030031 (10 μM). We obtained similar long-lasting facilitation in an in situ-perfused reparation from P11-P13 rats. On the basis of results from transection experiments of the rostral medulla and whole-cell recordings from preinspiratory neurons in the parafacial respiratory group (pFRG), we suggest that the rostral medulla, including the pFRG, is important to the induction of long-lasting facilitation. A histochemical analysis demonstrated a wide distribution of TRPA1 channel-positive cells in the reticular formation of the medulla, including the pFRG. Our findings suggest that TRPA1 channel activation could induce long-lasting facilitation of respiratory rhythm and provide grounds for future study on the roles of TRPA1 channels in the CNS., (Copyright © 2015 the American Physiological Society.)

Englerin A is a structurally unique natural product reported to selectively inhibit growth of renal cell carcinoma cell lines. A large scale phenotypic cell profiling experiment (CLiP) of englerin A on ¬over 500 well characterized cancer cell lines showed that englerin A inhibits growth of a subset of tumor cell lines from many lineages, not just renal cell carcinomas. Expression of the TRPC4 cation channel was the cell line feature that best correlated with sensitivity to englerin A, suggesting the hypothesis that TRPC4 is the efficacy target for englerin A. Genetic experiments demonstrate that TRPC4 expression is both necessary and sufficient for englerin A induced growth inhibition. Englerin A induces calcium influx and membrane depolarization in cells expressing high levels of TRPC4 or its close ortholog TRPC5. Electrophysiology experiments confirmed that englerin A is a TRPC4 agonist. Both the englerin A induced current and the englerin A induced growth inhibition can be blocked by the TRPC4/C5 inhibitor ML204. These experiments confirm that activation of TRPC4/C5 channels inhibits tumor cell line proliferation and confirms the TRPC4 target hypothesis generated by the cell line profiling. In selectivity assays englerin A weakly inhibits TRPA1, TRPV3/V4, and TRPM8 which suggests that englerin A may bind a common feature of TRP ion channels. In vivo experiments show that englerin A is lethal in rodents near doses needed to activate the TRPC4 channel. This toxicity suggests that englerin A itself is probably unsuitable for further drug development. However, since englerin A can be synthesized in the laboratory, it may be a useful chemical starting point to identify novel modulators of other TRP family channels.

Transient receptor potential (TRP) channels in the spinal dorsal horn lamina II (substantia gelatinosa; SG), which are involved in the modulation of nociceptive transmission, have not yet been fully examined in property. Activation of the TRP channels by various plant-derived chemicals results in an increase in the spontaneous release of L-glutamate onto the SG neurons. We examined the effects of a monoterpene ketone (-)-carvone (contained in spearmint) and its stereoisomer (+)-carvone (in caraway) on glutamatergic spontaneous excitatory transmission in SG neurons of adult rat spinal cord slices by using the whole-cell patch-clamp technique. (-)-Carvone and (+)-carvone increased the frequency of spontaneous excitatory postsynaptic current (sEPSC) in a reversible and concentration-dependent manner with a small increase in its amplitude. Half-maximal effective concentrations of (-)-carvone and (+)-carvone in increasing sEPSC frequency were 0.70 mM and 0.72 mM, respectively. The (-)-carvone but not (+)-carvone activity was inhibited by a TRPV1 antagonist capsazepine. On the other hand, the (+)-carvone but not (-)-carvone activity was inhibited by a TRPA1 antagonist HC-030031. These results indicate that (-)-carvone and (+)-carvone activate TRPV1 and TRPA1 channels, respectively, resulting in an increase in spontaneous L-glutamate release onto SG neurons, with almost the same efficacy. Such a difference in TRP activation between the stereoisomers may serve to know the properties of TRP channels in the SG., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Dry eye (DE) disease is commonly associated with ocular surface inflammation, an unstable tear film and symptoms of irritation. However, little is known about the role of central neural mechanisms in DE. This study used a model for persistent aqueous tear deficiency, exorbital gland removal, to assess the effects of mustard oil (MO), a transient receptor potential ankyrin (TRPA1) agonist, on eyeblink and eyewipe behavior and Fos-like immunoreactivity (Fos-LI) in the trigeminal brainstem of male rats. Spontaneous tear secretion was reduced by about 50% and spontaneous eyeblinks were increased more than 100% in DE rats compared to sham rats. MO (0.02-0.2%) caused dose-related increases in eyeblink and forelimb eyewipe behavior in DE and sham rats. Exorbital gland removal alone was sufficient to increase Fos-LI at the ventrolateral pole of trigeminal interpolaris/caudalis (Vi/Vc) transition region, but not at more caudal regions of the trigeminal brainstem. Under barbiturate anesthesia ocular surface application of MO (2-20%) produced Fos-LI in the Vi/Vc transition, in the mid-portions of Vc and in the trigeminal caudalis/upper cervical spinal cord (Vc/C1) region that was significantly greater in DE rats than in sham controls. MO caused an increase in Fos-LI ipsilaterally in superficial laminae at the mid-Vc and Vc/C1 regions in a dose-dependent manner. Smaller, but significant, increases in Fos-LI also were seen in the contralateral Vc/C1 region in DE rats. TRPA1 protein levels in trigeminal ganglia from DE rats ipsilateral and contralateral to gland removal were similar. Persistent tear reduction enhanced the behavioral and trigeminal brainstem neural responses to ocular surface stimulation by MO. These results suggested that TRPA1 mechanisms play a significant role in the sensitization of ocular-responsive trigeminal brainstem neurons in this model for tear deficient DE., (Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.)

Aim: TRPC3 is a non-selective cation channel, which forms a Ca2+ entry pathway involved in cardiac remodelling. Our aim was to analyse acute electrophysiological and contractile consequences of TRPC3 activation in the heart., Methods and Results: We used a murine model of cardiac TRPC3 overexpression and a novel TRPC3 agonist, GSK1702934A, to uncover (patho)physiological functions of TRPC3. GSK1702934A induced a transient, non-selective conductance and prolonged action potentials in TRPC3-overexpressing myocytes but lacked significant electrophysiological effects in wild-type myocytes. GSK1702934A transiently enhanced contractility and evoked arrhythmias in isolated Langendorff hearts from TRPC3-overexpressing but not wild-type mice. Interestingly, pro-arrhythmic effects outlasted TRPC3 current activation, were prevented by enhanced intracellular Ca2+ buffering, and suppressed by the NCX inhibitor 3',4'-dichlorobenzamil hydrochloride. GSK1702934A substantially promoted NCX currents in TRPC3-overexpressing myocytes. The TRPC3-dependent electrophysiologic, pro-arrhythmic, and inotropic actions of GSK1702934A were mimicked by angiotensin II (AngII). Immunocytochemistry demonstrated colocalization of TRPC3 with NCX1 and disruption of local interaction upon channel activation by either GSK1702934A or AngII., Conclusion: Cardiac TRPC3 mediates Ca2+ and Na+ entry in proximity of NCX1, thereby elevating cellular Ca2+ levels and contractility. Excessive activation of TRPC3 is associated with transient cellular Ca2+ overload, spatial uncoupling between TRPC3 and NCX1, and arrhythmogenesis. We propose TRPC3-NCX micro/nanodomain communication as determinant of cardiac contractility and susceptibility to arrhythmogenic stimuli., (© The Author 2015. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Current therapies for common types of cancer such as renal cell cancer are often ineffective and unspecific, and novel pharmacological targets and approaches are in high demand. Here we show the unexpected possibility for the rapid and selective killing of renal cancer cells through activation of calcium-permeable nonselective transient receptor potential canonical (TRPC) calcium channels by the sesquiterpene (-)-englerin A. This compound was found to be a highly efficient, fast-acting, potent, selective, and direct stimulator of TRPC4 and TRPC5 channels. TRPC4/5 activation through a high-affinity extracellular (-)-englerin A binding site may open up novel opportunities for drug discovery aimed at renal cancer., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Introduction and Hypothesis: The aim of this study was to analyze the mechanism underlying cross-sensitization between the colon and the bladder via activation of transient receptor potential A1 (TRPA1) channels., Methods: Using female Sprague-Dawley rats, polyethylene catheters were inserted into the colon between two ligations at the levels of 40 and 60 mm rostral to the anus and into the bladder. (1) We examined changes in colon and bladder activity after the application of allyl isothiocyanate (AI, 50 mM, 300 μl), a TRPA1 activator, into the colon or the bladder in an awake condition. Inhibitory effects of the pretreatment with HC-030031 (HC, 3 mg/kg), a TRPA1 inhibitor, on colon-to-bladder cross-sensitization induced by AI instilled in the colon were also investigated. (2) We examined Evans blue (EB) dye extravasation after TRPA1 stimulation in the colon or the bladder to evaluate vascular permeability due to tissue inflammation., Results: (1) Intercontraction intervals during continuous saline infusion into the bladder (0.04 ml/min) were significantly decreased after the intracolonic AI application, which significantly increased mean intracolonic pressure, indicative of colon-to-bladder cross-sensitization. The AI-induced colon-to-bladder cross-sensitization was completely prevented by the pretreatment with intravenous application of HC. On the other hand, mean intracolonic pressure was significantly decreased after the intravesical AI application, which significantly increased mean intravesical pressure. (2) EB dye extravasation was significantly increased in the AI-treated inflamed organs and also in the bladder following intracolonic AI treatment., Conclusions: Colon-to-bladder cross-sensitization is mediated via TRPA1 stimulation in the colon, although TRPA1 expressed in the bladder does not seem to participate in bladder-to-colon cross-sensitization.

Eugenol is a bioactive plant extract used as an analgesic agent in dentistry. The structural similarity of eugenol to cinnamaldehyde, an active ligand for transient receptor potential ankyrin 1 (TRPA1), suggests that eugenol might produce its effect via TRPA1, in addition to TRPV1 as we reported previously. In this study, we investigated the effect of eugenol on TRPA1, by fura-2-based calcium imaging and patch clamp recording in trigeminal ganglion neurons and in a heterologous expression system. As the result, eugenol induced robust calcium responses in rat trigeminal ganglion neurons that responded to a specific TRPA1 agonist, allyl isothiocyanate (AITC), and not to capsaicin. Capsazepine, a TRPV1 antagonist failed to inhibit eugenol-induced calcium responses in AITC-responding neurons. In addition, eugenol response was observed in trigeminal ganglion neurons from TRPV1 knockout mice and human embryonic kidney 293 cell lines that express human TRPA1, which was inhibited by TRPA1-specific antagonist HC-030031. Eugenol-evoked TRPA1 single channel activity and eugenol-induced TRPA1 currents were dose-dependent with EC50 of 261.5μM. In summary, these results demonstrate that the activation of TRPA1 might account for another molecular mechanism underlying the pharmacological action of eugenol., (Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.)

Constipation is a major gastrointestinal motility disorder with clinical need for effective drugs. We previously reported that transient receptor potential ankyrin 1 (TRPA1) is highly expressed in enterochromaffin (EC) cells, which are 5-hydroxytryptamine (5-HT)-releasing cells, and might therefore be a novel target for constipation. Here, we examined the effects of ASP7663, a novel and selective TRPA1 agonist, in constipation models as well as an abdominal pain model. ASP7663 activated human, rat, and mouse TRPA1 and released 5-HT from QGP-1 cells, and oral but not intravenous administration of ASP7663 significantly improved the loperamide-induced delay in colonic transit in mice. While pretreatment with the TRPA1 antagonist HC-030031 and vagotomy both inhibited the ameliorating effect of oral ASP7663 on the colonic transit, both orally and intravenously administered ASP7663 significantly inhibited colorectal distension (CRD)-induced abdominal pain response in rats. Taken together, these results demonstrate that ASP7663 exerts both anti-constipation and anti-abdominal pain actions, the former is likely triggered from the mucosal side of the gut wall via activation of vagus nerves while the latter is assumed to be provoked through systemic blood flow. We conclude that ASP7663 can be an effective anti-constipation drug with abdominal analgesic effect., (© 2013 Published by Elsevier B.V.)

Nitro-oleic acid (OA-NO2), an electrophilic fatty acid nitroalkene byproduct of redox reactions, activates transient receptor potential ion channels (TRPA1 and TRPV1) in primary sensory neurons. To test the possibility that signaling actions of OA-NO2 might modulate TRP channels, we examined: (1) interactions between OA-NO2 and other agonists for TRPA1 (allyl-isothiocyanate, AITC) and TRPV1 (capsaicin) in rat dissociated dorsal root ganglion cells using Ca(2+) imaging and patch clamp techniques and (2) interactions between these agents on sensory nerves in the rat hindpaw. Ca(2+) imaging revealed that brief application (15-30 s) of each of the three agonists induced homologous desensitization. Heterologous desensitization also occurred when one agonist was applied prior to another agonist. OA-NO2 was more effective in desensitizing the response to AITC than the response to capsaicin. Prolonged exposure to OA-NO2 (20 min) had a similar desensitizing effect on AITC or capsaicin. Homologous and heterologous desensitizations were also demonstrated with patch clamp recording. Deltamethrin, a phosphatase inhibitor, reduced the capsaicin or AITC induced desensitization of OA-NO2 but did not suppress the OA-NO2 induced desensitization of AITC or capsaicin, indicating that heterologous desensitization induced by either capsaicin or AITC occurs by a different mechanism than the desensitization produced by OA-NO2. Subcutaneous injection of OA-NO2 (2.5mM, 35 μl) into a rat hindpaw induced delayed and prolonged nociceptive behavior. Homologous desensitization occurred with AITC and capsaicin when applied at 15 minute intervals, but did not occur with OA-NO2 when applied at a 30 min interval. Pretreatment with OA-NO2 reduced AITC-evoked nociceptive behaviors but did not alter capsaicin responses. These results raise the possibility that OA-NO2 might be useful clinically to reduce neurogenic inflammation and certain types of painful sensations by desensitizing TRPA1 expressing nociceptive afferents., (Published by Elsevier Inc.)

Tetrahydrohyperforin (IDN5706) is a semi-synthetic compound derived from hyperforin (IDN5522) and is the main active principle of St. John's Wort. IDN5706 has shown numerous beneficial effects when administered to wild-type and double transgenic (APPswe/PSEN1ΔE9) mice that model Alzheimer's disease. However, its mechanism of action is currently unknown. Toward this end, we analysed field excitatory postsynaptic potentials (fEPSPs) in mouse hippocampal slices incubated with IDN5706 and in the presence of the TRPC3/6/7 activator 1-oleoyl-2-acetyl-sn-glycerol (OAG), the TRPC channel blocker SKF96365, and neurotoxic amyloid β-protein (Aβ) oligomers. To study spatial memory, Morris water maze (MWM) behavioural tests were conducted on wild-type mice treated with IDN5706 and SKF96365. In silico studies were conducted to predict a potential pharmacophore. IDN5706 and OAG had a similar stimulating effect on fEPSPs, which was inhibited by SKF96365. IDN5706 protected from reduced fEPSPs induced by Aβ oligomers. IDN5706 improved spatial memory in wild-type mice, an effect that was counteracted by co-administration of SKF96365. Our in silico studies suggest strong pharmacophore similarity of IDN5706 and other reported TRPC6 activators (IDN5522, OAG and Hyp9). We propose that the effect of IDN5706 is mediated through activation of the TRPC3/6/7 channel subfamily. The unveiling of the drug's mechanism of action is a necessary step toward the clinical use of IDN5706 in Alzheimer's disease.

Background and Purpose: The transient receptor potential channel C5 (TRPC5) is a Ca(2+)-permeable cation channel, which is predominantly expressed in the brain. TRPC5 is activated in a PLC-dependent manner by, as yet, unidentified endogenous messengers. Recently, modulators of TRPC5, like Ca(2+), pH and phospholipids, have been identified. However, the role of TRPC5 in vivo is only poorly understood. Novel specific modulators of TRPC5 might help to elucidate its function., Experimental Approach: Novel modulators of TRPC5 were identified in a compound screening of approved drugs and natural compounds. The potency and selectivity of TRPC5-activating compounds were determined by fluorometric calcium imaging. The biophysical properties of channel activation by these compounds were analysed using electrophysiological measurements., Key Results: Riluzole was identified as a novel activator of TRPC5 (EC₅₀ 9.2 ± 0.5 μM) and its mechanism of action was shown to be independent of G protein signalling and PLC activity. Riluzole-induced TRPC5 currents were potentiated by La(3+) and, utilizing TRPC5 mutants that lack La(3+) binding sites, it was confirmed that riluzole and La(3+) activate TRPC5 by different mechanisms. Recordings of excised inside-out patches revealed a relatively direct effect of riluzole on TRPC5., Conclusions and Implications: Riluzole can activate TRPC5 heterologously expressed in HEK293 cells as well as those endogenously expressed in the U-87 glioblastoma cell line. Riluzole does not activate any other member of the TRPC family and could, therefore, despite its action on other ion channels, be a useful pharmacological tool for identifying TRPC5-specific currents in immortalized cell lines or in acutely isolated primary cells., (© 2013 The British Pharmacological Society.)

Objective: Gout is a common cause of inflammatory arthritis and is provoked by the accumulation of monosodium urate (MSU) crystals. However, the underlying mechanisms of the pain associated with acute attacks of gout are poorly understood. The aim of this study was to evaluate the role of transient receptor potential ankyrin 1 (TRPA-1) and TRPA-1 stimulants, such as H2 O2 , in a rodent model of MSU-induced inflammation., Methods: MSU or H2 O2 was injected into the hind paws of rodents or applied in cultured sensory neurons, and the intracellular calcium response was measured in vitro. Inflammatory or nociceptive responses in vivo were evaluated using pharmacologic, genetic, or biochemical tools and methods., Results: TRPA-1 antagonism, TRPA-1 gene deletion, or pretreatment of peptidergic TRP-expressing primary sensory neurons with capsaicin markedly decreased MSU-induced nociception and edema. In addition to these neurogenic effects, MSU increased H2 O2 levels in the injected tissue, an effect that was abolished by the H2 O2 -detoxifying enzyme catalase. H2 O2 , but not MSU, directly stimulated sensory neurons through the activation of TRPA-1. The nociceptive responses evoked by MSU or H2 O2 injection were attenuated by the reducing agent dithiothreitol. In addition, MSU injection increased the expression of TRPA-1 and TRP vanilloid channel 1 (TRPV-1) and also enhanced cellular infiltration and interleukin-1β levels, and these effects were blocked by TRPA-1 antagonism., Conclusion: Our results suggest that MSU injection increases tissue H2 O2 , thereby stimulating TRPA-1 on sensory nerve endings to produce inflammation and nociception. TRPV-1, by a previously unknown mechanism, also contributes to these responses., (Copyright © 2013 by the American College of Rheumatology.)

Following the recent identification of the naturally occurring 3-ylidene-4,5-dihydrophthalide ligustilide and its oxidation product dehydroligustilide as novel TRPA1 modulators, a series of seventeen 3-ylidenephthalides was synthesized and tested on TRPA1 and TRPM8 channels. Most of these compounds acted as strong modulators of the two channel types with EC50 and/or IC50 values distinctly lower than those of the reference compounds., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The sensitization of capsaicin-sensitive lung vagal (CSLV) afferents by inflammatory mediators is important in the development of airway hypersensitivity. Hydrogen sulfide (H2S) is an endogenous mediator inducing hyperalgesia through transient receptor potential ankyrin 1 (TRPA1) receptors located on nociceptors. We conducted this study to determine whether H2S elevates the sensitivity of rat CSLV afferents. In anesthetized, artificially ventilated rats, the inhalation of aerosolized sodium hydrosulfide (NaHS, a H2S donor) caused no significant changes in the baseline activity of CSLV afferents. However, the afferent responses to right atrial injection of capsaicin or phenylbiguanide and to lung inflation were all markedly potentiated after NaHS inhalation. By contrast, the inhalation of its vehicle or NaOH (with a similar pH to NaHS) failed to enhance the afferent responses. Additionally, the potentiating effect on the afferent responses was found in rats inhaling L-cysteine (a substrate of H2S synthase) that slowly releases H2S. The potentiating effect of NaHS on the sensitivity of CSLV afferents was completely blocked by pretreatment of HC-030031 (a TRPA1 receptor antagonist) but was unaffected by its vehicle. In isolated rat CSLV neurons, the perfusion of NaHS alone did not influence the intracellular Ca(2+) concentration but markedly potentiated the Ca(2+) transients evoked by capsaicin. The NaHS-caused effect was totally abolished by HC-030031 pretreatment. These results suggest that H2S induces a nonspecific sensitizing effect on CSLV fibers to both chemical and mechanical stimulation in rat lungs, which appears mediated through an action on the TRPA1 receptors expressed on the nerve endings of CSLV afferents.

Both TRPV1 and TRPA1 are non-selective cation channels. They are co-expressed, and interact in sensory neurons such as dorsal root ganglia (DRG) and trigeminal ganglia (TG), and are involved in nociception, being activated by nociceptive stimuli. Immunohistological localization of TRPV1 in vestibular ganglion (VG) neurons has been reported. Although TRPA1 is co-expressed with TRPV1 in DRG and TG neurons, it is unclear whether TRPA1 channels are expressed in VG neurons. Moreover, it is unknown whether TRPV1 and TRPA1 channels are functional in VG neurons. We investigated the expression of TRPV1 and TRPA1 in rat VG neurons by RT-PCR, in situ hybridization, immunohistochemistry, and Ca(2+) imaging experiments. Both TRPV1 and TRPA1 RT-PCR products were amplified from the mRNA of rat VG neurons. In situ hybridization experiments showed TRPV1 and TRPA1 mRNA expression in the majority of VG neurons. Immunohistochemistry experiments confirmed TRPV1 protein expression. In Ca(2+) imaging experiments, capsaicin, a TRPV1 agonist, induced a significant increase in intracellular calcium ion concentration ([Ca(2+)]i) in rat primary cultured VG neurons, which was almost completely blocked by capsazepine, a TRPV1-specific antagonist. Cinnamaldehyde, a TRPA1 agonist, also caused an increase in [Ca(2+)]i, which was completely inhibited by HC030031, a TRPA1-specific antagonist. Moreover, in some VG neurons, a [Ca(2+)]i increase was evoked by both capsaicin and cinnamaldehyde in the same neuron. In summary, our histological and physiological studies reveal that TRPV1 and TRPA1 are expressed in VG neurons. It is suggested that TRPV1 and TRPA1 in VG neurons might participate in vestibular function and/or dysfunction such as vertigo., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Abdominal pain is a frequent symptom of peritoneal cavity irritation, but little is known about the role of the receptors for irritant substances, transient receptor potential vanilloid 1 (TRPV1) and ankyrin 1 (TRPA1), in this painful condition. Thus, we investigated the abdominal nociception caused by peritoneal stimulation with TRPV1 (capsaicin) and TRPA1 (allyl isothiocyanate, AITC) agonists and their mechanisms in rats. The intraperitoneal (i.p.) injection of either capsaicin or AITC (0.03-10 mg/kg) induced short-term (up to 20 min) and dose-dependent abdominal nociception, and also produced c-fos expression in spinal afferents of the dorsal horn. TRPV1 antagonism prevented (94 ± 4% inhibition) nociception induced by capsaicin but not by AITC. In contrast, the TRPA1 antagonism almost abolished AITC-induced nociception (95 ± 2% inhibition) without altering the capsaicin response. Moreover, nociception induced by either capsaicin or AITC was reduced by the desensitisation of TRPV1-positive sensory fibres with resiniferatoxin (73 ± 18 and 76 ± 15% inhibitions, respectively) and by the NK1 receptor antagonist aprepitant (56 ± 5 and 53 ± 8% inhibitions, respectively). Likewise, the i.p. injections of capsaicin or AITC increased the content of substance P in the peritoneal fluid. Nevertheless, neither the mast cell membrane stabiliser cromoglycate, nor the H1 antagonist promethazine, nor depletion of peritoneal macrophages affected abdominal nociception induced either by capsaicin or AITC. Accordingly, neither capsaicin nor AITC increased the histamine content in the peritoneal fluid or provoked peritoneal mast cell degranulation in vitro. Collectively, our findings suggest that TRPV1 and TRPA1 stimulation in the peritoneum produces abdominal nociception that is mediated by sensory fibres activation., (© 2013 Elsevier B.V. All rights reserved.)

Transient receptor potential (TRP) channels are thought to play a role in regulating nociceptive transmission to spinal substantia gelatinosa (SG) neurons. It remains to be unveiled whether the TRP channels in the central nervous system are different in property from those involved in receiving nociceptive stimuli in the peripheral nervous system. We examined the effect of the vanilloid compound zingerone, which activates TRPV1 channels in the cell body of a primary afferent neuron, on glutamatergic excitatory transmission in the SG neurons of adult rat spinal cord slices by using the whole cell patch-clamp technique. Bath-applied zingerone reversibly and concentration-dependently increased spontaneous excitatory postsynaptic current (EPSC) frequency. This effect was accompanied by an inward current at -70 mV that was resistant to glutamate receptor antagonists. These zingerone effects were repeated and persisted in Na(+)-channel blocker tetrodotoxin-, La(3+)-, or IP3-induced Ca(2+)-release inhibitor 2-aminoethoxydiphenyl borate-containing or Ca(2+)-free Krebs solution. Zingerone activity was resistant to the selective TRPV1 antagonist capsazepine but sensitive to the nonselective TRP antagonist ruthenium red, the TRPA1 antagonist HC-030031, and the Ca(2+)-induced Ca(2+)-release inhibitor dantrolene. TRPA1 agonist allyl isothiocyanate but not capsaicin inhibited the facilitatory effect of zingerone. On the other hand, zingerone reduced monosynaptically evoked EPSC amplitudes, as did TRPA1 agonists. Like allyl isothiocyanate, zingerone enhanced GABAergic spontaneous inhibitory transmission in a manner sensitive to tetrodotoxin. We conclude that zingerone presynaptically facilitates spontaneous excitatory transmission, probably through Ca(2+)-induced Ca(2+)-release mechanisms, and produces a membrane depolarization in SG neurons by activating TRPA1 but not TRPV1 channels.

We characterized pain behavior and cutaneous blood flow response induced by activation of the spinal transient receptor potential ankyrin 1 (TRPA1) channel using intrathecal drug administrations in the rat. Additionally, we assessed whether the pronociceptive actions induced by intrathecally administered dynorphin A, cholecystokinin or prostaglandin F(2α) are mediated by the spinal TRPA1 channel. Cinnamaldehyde, a TRPA1 agonist, produced a dose-related (3-10 μg) cutaneous blood flow increase and mechanical hypersensitivity effect. These effects at the currently used doses were of short duration and attenuated, although not completely, by pretreatment with A-967079, a TRPA1 antagonist. The cinnamaldehyde-induced hypersensitivity was also reduced by pretreatment with minocycline (an inhibitor of microglial activation), but not by carbenoxolone (a gap junction decoupler). In vitro study, however, indicated that minocycline only poorly blocks the TRPA1 channel. The mechanical hypersensitivity effect induced by dynorphin A, but not that by cholecystokinin or prostaglandin F(2α), was attenuated by a TRPA1 antagonist Chembridge-5861528 as well as A-967079. The cinnamaldehyde-induced cutaneous blood flow increase was not suppressed by MK-801, an NMDA receptor antagonist, or bicuculline, a GABA(A) receptor antagonist. The results indicate that spinal TRPA1 channels promote mechanical pain hypersensitivity and due to antidromic activation of nociceptive nerve fibers increase cutaneous blood flow. The attenuation of the cinnamaldehyde-induced hypersensitivity effect by minocycline may be explained by action other than block of the TRPA1 channel. Moreover, the spinal TRPA1 channel is involved in mediating the pronociceptive action of dynorphin A, but not that of the spinal cholecystokinin or prostaglandin F(2α).

Accumulating evidence shows that hydrogen sulfide (H2S) has a variety of physiological functions. H2S is produced from cysteine by 3 sulfurtransferases. H2S, in turn, generates polysulfides, the functions of which are not well understood. H2S induces Ca(2+) influx in astrocytes, a type of glia. However, the receptor that mediates the response has not been identified. Here, we have shown that polysulfides induce Ca(2+) influx by activating transient receptor potential (TRP)A1 channels in rat astrocytes (EC50 91 nM, Hill coefficient value 1.77±0.26) and that the maximum response was induced at 0.5 μM, which is 1/320 of the concentration of H2S required to achieve a response of similar magnitude (160 μM, EC50 116 μM). TRPA1-selective agonists, allyl isothiocyanate and cinnamaldehyde, induced Ca(2+) influx, and responses to polysulfides were suppressed by TRPA1-selective inhibitors, HC-030031 and AP-18, as well as by siRNAs selective to TRPA1. The present study suggests that polysulfides are possible H2S-derived signaling molecules that stimulate TRP channels in the brain.

The functional roles of transient receptor potential (TRP) channels in the gastrointestinal tract have garnered considerable attention in recent years. We previously reported that daikenchuto (TU-100), a traditional Japanese herbal medicine, increased intestinal blood flow (IBF) via adrenomedullin (ADM) release from intestinal epithelial (IE) cells (Kono T et al. J Crohns Colitis 4: 161-170, 2010). TU-100 contains multiple TRP activators. In the present study, therefore, we examined the involvement of TRP channels in the ADM-mediated vasodilatatory effect of TU-100. Rats were treated intraduodenally with the TRP vanilloid type 1 (TRPV1) agonist capsaicin (CAP), the TRP ankyrin 1 (TRPA1) agonist allyl-isothiocyanate (AITC), or TU-100, and jejunum IBF was evaluated using laser-Doppler blood flowmetry. All three compounds resulted in vasodilatation, and the vasodilatory effect of TU-100 was abolished by a TRPA1 antagonist but not by a TRPV1 antagonist. Vasodilatation induced by AITC and TU-100 was abrogated by anti-ADM antibody treatment. RT-PCR and flow cytometry revealed that an IEC-6 cell line originated from the small intestine and purified IE cells expressed ADM and TRPA1 but not TRPV1. AITC increased ADM release in IEC cells remarkably, while CAP had no effect. TU-100 and its ingredient 6-shogaol (6SG) increased ADM release dose-dependently, and the effects were abrogated by a TRPA1 antagonist. 6SG showed similar TRPA1-dependent vasodilatation in vivo. These results indicate that TRPA1 in IE cells may play an important role in controlling bowel microcirculation via ADM release. Epithelial TRPA1 appears to be a promising target for the development of novel strategies for the treatment of various gastrointestinal disorders.

TRPC1 is a major component of store-operated calcium entry in many cell types. In our previous studies, three types of endogenous store-operated calcium channels have been described in HEK293 cells, but it remained unknown which of these channels are composed of TRPC1 proteins. Here, this issue has been addressed by performing single-channel analysis in HEK293 cells transfected with anti-TRPC1 siRNA (siTPRC1) or a TPRC1-encoding plasmid. The results show that thapsigargin-or agonist-induced calcium influx is significantly attenuated in siTRPC1-transfected HEK293 cells. TRPC1 knockdown by siRNA results in the disappearance of store-operated I(max) channels, while the properties of I(min) and I(NS) channels are unaffected. In HEK293 cells with overexpressed TRPC1 protein, the unitary current-voltage relationship of exogenous TRPC1 channels is almost linear, with a slope conductance of about 17 pS. The extrapolated reversal potential of expressed TRPC1 channels is +30 mV. Therefore, the main electrophysiological and regulatory properties of expressed TRPC1 and native I(max) channels are identical. Moreover, TRPC1 overexpression in HEK293 cells results in an increased number of store-operated I(max) channels. All these data allow us to conclude that TRPC1 protein forms native store-operated I(max) channels but is not an essential subunit for other store-operated channel types in HEK293 cells., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

Objective: Stimulation of endothelial TRP channels, specifically TRPA1, promotes vasodilation of cerebral arteries through activation of Ca2+ -dependent effectors along the myoendothelial interface. However, presumed TRPA1-triggered endothelial Ca2+ signals have not been described. We investigated whether TRPA1 activation induces specific spatial and temporal changes in Ca2+ signals along the intima that correlates with incremental vasodilation., Methods: Confocal imaging, immunofluorescence staining, and custom image analysis were employed., Results: We found that endothelial cells of rat cerebral arteries exhibit widespread basal Ca2+ dynamics (44 ± 6 events/minute from 26 ± 3 distinct sites in a 3.6 × 10(4) μm2 field). The TRPA1 activator AITC increased Ca2+ signals in a concentration-dependent manner, soliciting new events at distinct sites. Origination of these new events corresponded spatially with TRPA1 densities in IEL holes, and the events were prevented by the TRPA1 inhibitor HC-030031. Concentration-dependent expansion of Ca2+ events in response to AITC correlated precisely with dilation of pressurized cerebral arteries (p = 0.93 by F-test). Correspondingly, AITC caused rapid endothelium-dependent suppression of asynchronous Ca2+ waves in subintimal smooth muscle., Conclusions: Our findings indicate that factors that stimulate TRPA1 channels expand Ca2+ signal-effector coupling at discrete sites along the endothelium to evoke graded cerebral artery vasodilation., (© 2012 John Wiley & Sons Ltd.)

The standardized extract of the St. John's wort plant (Hypericum perforatum) is commonly used to treat mild to moderate depression. Its active constituent is hyperforin, a phloroglucinol derivative that reduces the reuptake of serotonin and norepinephrine by increasing intracellular Na(+) concentration through the activation of nonselective cationic TRPC6 channels. TRPC6 channels are also Ca(2+) -permeable, resulting in intracellular Ca(2+) elevations. Indeed, hyperforin activates TRPC6-mediated currents and Ca(2+) transients in rat PC12 cells, which induce their differentiation, mimicking the neurotrophic effect of nerve growth factor. Here, we show that hyperforin modulates dendritic spine morphology in CA1 and CA3 pyramidal neurons of hippocampal slice cultures through the activation of TRPC6 channels. Hyperforin also evoked intracellular Ca(2+) transients and depolarizing inward currents sensitive to the TRPC channel blocker La(3+) , thus resembling the actions of the neurotrophin brain-derived neurotrophic factor (BDNF) in hippocampal pyramidal neurons. These results suggest that the antidepressant actions of St. John's wort are mediated by a mechanism similar to that engaged by BDNF., (Copyright © 2012 Wiley Periodicals, Inc.)

The transient receptor potential ankyrin 1 (TRPA1) is expressed in peripheral and spinal terminals of sensory neurons, jointly to the vanilloid receptor (TRPV1). A relevant peripheral role of TRPA1 receptor has been implicated in a variety of processes, including the detection of noxious cold, and diverse painful stimulus, but the functional role of TRPA1 receptor in nociceptive transmission at spinal cord in vivo is poorly known. Therefore, the aim of this study was to evaluate whether the glutamatergic system is involved in the transmission of nociceptive stimulus induced for a TRPA1 agonist in the rat spinal cord. We observed that cinnamaldehyde, a TRPA1 agonist, on spinal cord synaptosomes leads to an increase in [Ca(2+)](i) and a rapid release of glutamate, but was not able to change the specific [(3)H]-glutamate binding. In addition, spinally administered cinnamaldehyde produced heat hyperalgesia and mechanical allodynia in rats. This behavior was reduced by the co-injection (i.t.) of camphor (TRPA1 antagonist) or MK-801 (N-methyl-D-aspartate (NMDA) receptor antagonist) to cinnamaldehyde. Besides, the pretreatment with resiniferatoxin (RTX), a potent TRPV1 agonist, abolished the cinnamaldehyde-induced heat hyperalgesia. Here, we showed that intrathecal RTX results in a decrease in TRPA1 and TRPV1 immunoreactivity in dorsal root ganglion. Collectively, our results demonstrate the pertinent participation of spinal TRPA1 in the possible enhancement of glutamatergic transmission of nociceptive signals leading to increase of the hypersensitivity, here observed as heat hyperalgesia. Then the modulation of spinal TRPA1 might be a valuable target in painful conditions associated with central pain hypersensitivity., (Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.)

Background: Propofol-evoked injection site pain is not observed with fospropofol. We hypothesized that unlike propofol, fospropofol does not activate the irritant receptor, transient receptor potential 1 (TRPA1)., Methods: We tested the hypothesis using electrophysiology and behavioral studies., Results: Our data demonstrate that propofol (100 μM) evokes an inward current only in TRPA1-expressing neurons. However, fospropofol (100 μM and 1 mM) is unable to evoke depolarizing currents in either TRPA1-positive or TRPA1-negative neurons. Both propofol and fospropofol produced general anesthesia., Conclusions: The lack of algogenic activity in fospropofol is most likely the result of its inability to activate TRPA1 on nociceptors.

Activation of transient receptor potential ankyrin-1 (TRPA1) on meningeal nerve endings has been suggested to contribute to environmental irritant-induced headache, but this channel may also contribute to other forms of headache, such as migraine. The preclinical studies described here examined functional expression of TRPA1 on dural afferents and investigated whether activation of TRPA1 contributes to headache-like behaviors. Whole-cell patch-clamp recordings were performed in vitro with 2 TRPA1 agonists, mustard oil (MO), and the environmental irritant umbellulone (UMB) on dural-projecting trigeminal ganglion neurons. Application of MO and UMB to dural afferents produced TRPA1-like currents in approximately 42% and 38% of cells, respectively. By means of an established in vivo behavioral model of migraine-related allodynia, dural application of MO and UMB produced robust time-related tactile facial and hind paw allodynia that was attenuated by pretreatment with the TRPA1 antagonist HC-030031. Additionally, MO or UMB were applied to the dura, and exploratory activity was monitored for 30min with an automated open-field activity chamber. Dural MO and UMB decreased the number of vertical rearing episodes and the time spent rearing in comparison to vehicle-treated animals. This change in activity was prevented in rats pretreated with HC-030031 as well as sumatriptan, a clinically effective antimigraine agent. These data indicate that TRPA1 is expressed on a substantial fraction of dural afferents, and activation of meningeal TRPA1 produces behaviors consistent with those observed in patients during migraine attacks. Further, they suggest that activation of meningeal TRPA1 via endogenous or exogenous mechanisms can lead to afferent signaling and headache., (Copyright © 2012 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.)

Cholecystokinin (CCK) is one of the most abundant neuropeptides in the brain where it interacts with two G protein-coupled receptors (CCK1 and CCK2). Both types of CCK receptors are coupled to G(q/11) proteins resulting in increased function of phospholipase C (PLC) pathway. Whereas CCK has been suggested to increase neuronal excitability in the brain via activation of cationic channels, the types of cationic channels have not yet been identified. Here, we co-expressed CCK2 receptors and TRPC5 channels in human embryonic kidney (HEK) 293 cells and studied the effects of CCK on TRPC5 channels using patch-clamp techniques. Our results demonstrate that activation of CCK2 receptors robustly potentiates the function of TRPC5 channels. CCK-induced activation of TRPC5 channels requires the functions of G-proteins and PLC and depends on extracellular Ca(2+). The activation of TRPC5 channels mediated by CCK2 receptors is independent of IP(3) receptors and protein kinase C. CCK-induced opening of TRPC5 channels is not store-operated because application of thapsigargin to deplete intracellular Ca(2+) stores failed to alter CCK-induced TRPC5 channel currents significantly. Bath application of CCK also significantly increased the open probability of TRPC5 single channel currents in cell-attached patches. Because CCK exerts extensive effects in the brain, our results may provide a novel mechanism to explain its roles in modulating neuronal excitability., (Copyright © 2012 Elsevier B.V. All rights reserved.)