Differential behavioral effect of the TRPM8/TRPA1 channel agonist icilin (AG-3-5)

Molecular identification of two new transient receptor potential (TRP) channels, TRPM8 and TRPA1, has prompted an intense interest in their functional roles. We report that an acute exposure to the TRPM8/TRPA1 agonist icilin (0.01-100 microM), but not TRPV1 agonist capsaicin (10 microM), causes an atypical dose-related increase in planarian motility. This is the first demonstration of a TRPM8/TRPA1 channel subtype agonist-induced differential pharmacological effect in invertebrates and provides a novel sensitive, quantifiable end-point for studying TRP channel pharmacology.     

Lack of transient receptor potential melastatin 8 activation by phthalate esters that enhance contact hypersensitivity in mice

We studied the involvement of sensory neurons in skin sensitization to allergens using a mouse model in which the T-helper type 2 response is essential. Skin sensitization to fluorescein isothiocyanate (FITC) has been shown to be enhanced by several phthalate esters, including dibutyl phthalate (DBP). For different types of phthalate esters, we found a correlation between the ability of transient receptor potential (TRP) A1 activation and that of enhancing skin sensitization. A TRPA1-specific antagonist, HC-030031, was shown to suppress skin sensitization in the presence of DBP. However, since phthalate esters also activate TRPV1, phthalate esters could activate other types of TRP channels non-selectively. Furthermore, sensitization to FITC is also enhanced by menthol, which activates TRPA1 and TRPM8. Here we established an in vitro system for measuring TRPM8 activation. The selectivity for TRPM8 was established by the fact that two TRPM8 agonists (menthol and icilin) induced calcium mobilization, whereas agonists of TRPA1 and TRPV1 did not. We demonstrated that phthalate esters do not activate TRPM8. TRPA1-antagonist HC-030031 did not inhibit TRPM8 activation induced by menthol or icilin. These results show that phthalate esters activate TRPA1 and TRPV1 with selectivity. TRPM8 activation is not likely to be involved in the sensitization to FITC.     

Modulation of mouse gastrointestinal motility by allyl isothiocyanate, a constituent of cruciferous vegetables (Brassicaceae): evidence for TRPA1-independent effects

BACKGROUND AND PURPOSE

Allyl isothiocyanate (AITC, mustard oil), a constituent of many common cruciferous vegetables (Brassicaceae), activates transient receptor potential of ankyrin type-1 (TRPA1) channels, claimed to regulate gastrointestinal contractility. In this study, we have investigated the effect of AITC on intestinal motility.

EXPERIMENTAL APPROACH

Effects of AITC were investigated in vivo on upper gastrointestinal transit in mice and in mouse isolated ileum [contractions induced by electrical field stimulation (EFS), acetylcholine and spontaneous contractility]. The contractor activity of AITC was studied in mouse isolated colon. The ability of TRPA1 channel antagonists to block AITC-induced elevation of intracellular Ca²⁺[Ca²⁺]_i was assessed in HEK293 cells transfected with rat TRPA1 channels.

KEY RESULTS

AITC increased [Ca²⁺]_i in HEK293 cells, reduced ileal contractility (acetylcholine-, EFS-induced contractions and spontaneous contractility), but contracted the isolated colon. Gentamicin and camphor (non-selective TRPA1 channel antagonists), HC-030031 and AP18 (selective TRPA1 channel agonists) inhibited AITC-induced effects in HEK293 cells but not in the ileum or colon. AITC-induced contractions were reduced by tetrodotoxin and strongly reduced by nifedipine, cyclopiazonic acid and ryanodine. In vivo, AITC reduced (following i.p. administration) or increased (following intragastric administration) upper gastrointestinal transit in mice These effects were not affected by HC-030031.

CONCLUSION AND IMPLICATIONS

AITC, depending, in vitro, on the regions of gut examined and, in vivo, on the route of administration, exerted both stimulatory and inhibitory effects on intestinal motility, which were not sensitive to TRPA1 channel antagonists. The proposition that TRPA1 channels are the primary targets for AITC to induce contraction should be revised.     

Purinoceptor‐mediated,Capsaicin‐resistant Excitatory Effect of Allyl Isothiocyanate on Neurons of the Guinea‐Pig Small Intestine

Allyl isothiocyanate (AITC; 200 μM) caused atropine‐ and tetrodotoxin‐sensitive longitudinal muscle contraction on the guinea‐pig small intestine. The response was not influenced by hexamethonium, a functional blockade of capsaicin‐sensitive neurons or by antagonists acting at TRPV1 or TRPA1, but was abolished by the P₂ purinoceptor antagonist PPADS (50 μM). It is concluded that cholinergic motoneurons are activated by a purinergic mechanism in the course of the AITC response, independently of capsaicin‐sensitive processes or even TRPA1.     

Identification of herbal components as TRPA1 agonists and TRPM8 antagonists

Transient receptor potential (TRP) channels are non-selective cation channels that are implicated in analgesia, bowel motility, wound healing, thermoregulation, vasodilation and voiding dysfunction. Many natural products have been reported to affect the activity of TRP channels. We hypothesize that numerous traditional herbal medicines (THMs) might exert their pharmacological activity through modulating the activity of TRP channels. The present study aimed to evaluate the effects of flavonoid aglycones and their glycosides, which are the main components of many THMs, on the TRP channel subtypes. A Ca²⁺ influx assay was performed using recombinant human TRPA1, TRPV1, TRPV4 and TRPM8 cell lines. Our findings showed that flavonoid aglycones and glycycoumarin activated TRPA1. In particular, isoflavone and chalcone compounds displayed potent TRPA1 agonistic activity. Furthermore, flavone aglycones showed concomitant potent TRPM8 inhibiting activity. Indeed, flavone, isoflavone aglycones, non-prenylated chalcones and glycycoumarin were found to be TRPM8 inhibitors. Hence, flavonoid aglycones metabolized by lactase-phlorizin hydrolase and β-glucosidase in the small intestine or gut microbiota of the large intestine could generate TRPA1 agonists and TRPM8 antagonists.

     

Overexpression of human transient receptor potential M5 upregulates endogenous human transient receptor potential A1 in a stable HEK cell line

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

Comparison of TRPA1-versus TRPV1-mediated cough in guinea pigs

TRPA1 receptor is activated by endogenous inflammatory mediators and exogenous pollutant molecules relevant to respiratory diseases. Previous studies have implicated TRPA1 as a drug target for antitussive therapy. Here we evaluated the relative efficacy of TRPA1 activation to evoke cough. In conscious guinea pigs the TRPA1 agonist allyl-isothiocyanate (AITC) evoked cough with a maximally effective concentration of 10mM that was abolished by the selective TRPA1 antagonist AP-18. AITC (10mM) was approximately 3-times less effective in inducing cough than capsaicin (50 μM). Ex vivo single fiber extracellular recordings revealed that, similarly to capsaicin, AITC evoked activation in airway jugular C-fibers, but not in airway nodose Aδ-fibers. Consistent with the cough studies, AITC was approximately 3-times less effective than capsaicin in evoking sustained activation of the jugular C-fibers. Another TRPA1 agonist, cinnamaldehyde, was approximately twofold more effective than AITC in inducing cough. However, the cinnamaldehyde (10mM)-induced cough was only partially inhibited by the TRPA1 antagonist AP-18, and was abolished by combination of AP-18 and the TRPV1 antagonist I-RTX. We conclude that in na?ve guinea pigs, TRPA1 activation initiates cough that is relatively modest compared to the cough initiated by TRPV1, likely due to lower efficacy of TRPA1 stimulation to induce sustained activation of airway C-fibers.     

化疗引起的神经痛治疗靶点温度选择性TRP通道的研究进展

作为钙离子渗透性的瞬时受体电位(TRP),5种通道(TRPV1~4和TRPM2)被不同的高温激活,两种通道(TRPV1和TRPV8)被低温激活。越来越多的证据表明,TRPA1和TRPM8拮抗剂可预防顺铂、奥沙利铂和紫杉醇诱导的线粒体氧化应激、炎症、冷痛和痛觉过敏。TRPV1在顺铂引起的感觉神经元热痛觉和机械异常中有应答。TRPA1、TRPM8和TRPV2蛋白表达水平主要通过这些治疗方法在背根(DRG)和三叉神经节中增加。主要总结了5种温度调节TRP通道(TRPA1、TRPM8、TRPV1、TRPV2和TRPV4)。     

Contractile mechanisms coupled to TRPA1 receptor activation in rat urinary bladder

TRPA1 is a member of the transient receptor potential (TRP) channel family present in sensory neurons. Here we show that vanilloid receptor (TRPV1) stimulation with capsaicin and activation of TRPA1 with allyl isothiocyanate or cinnamaldehyde cause a graded contraction of the rat urinary bladder in vitro. Repeated applications of maximal concentrations of the agonists produce desensitization to their contractile effects. Moreover, contraction caused by TRPA1 agonists generates cross-desensitization with capsaicin. The TRP receptor antagonist ruthenium red (10-100 microM) inhibits capsaicin (0.03 microM), allyl isothiocyanate (100 microM) and cinnamaldehyde (300 microM)-induced contractions in the rat urinary bladder. The selective TRPV1 receptor antagonist SB 366791 (10 microM) blocks capsaicin-induced contraction, but partially reduces allyl isothiocyanate- or cinnamaldehyde-mediated contraction. However, allyl isothiocyanate and cinnamaldehyde (10-1000 microM) completely fail to interfere with the specific binding sites for the TRPV1 agonist [(3)H]-resiniferatoxin. Allyl isothiocyanate or cinnamaldehyde-mediated contractions of rat urinary bladder, which rely on external Ca(2+) influx, are significantly inhibited by tachykinin receptor antagonists as well as by tetrodotoxin (1 microM) or indomethacin (1 microM). Allyl isothiocyanate-induced contraction is not changed by atropine (1 microM) or suramin (300 microM). The exposure of urinary bladders to allyl isothiocyanate (100 microM) causes an increase in the prostaglandin E(2) and substance P levels. Taken together, these results indicate that TRPA1 agonists contract rat urinary bladder through sensory fibre stimulation, depending on extracellular Ca(2+) influx and release of tachykinins and cyclooxygenase metabolites, probably prostaglandin E(2). Thus, TRPA1 appears to exert an important role in urinary bladder function.     

Icilin induces a hyperthermia in rats that is dependent on nitric oxide production and NMDA receptor activation

Icilin (AG-3-5) is a cold-inducing agent that activates the transient receptor potential channels TRPM8 and TRPA1. Both channels are members of the transient receptor potential (TRP) superfamily of ion channels and are activated by cold. Despite the key role of cold-activated TRPM8 and TRPA1 channels in temperature sensation and other physiological processes, the significance of these channels in thermoregulation in conscious animals is poorly understood. Therefore, in the present study we investigated the effects of icilin on body temperature in rats and tested the hypothesis that cold-activated TRP channel activation by icilin causes a hyperthermia which requires nitric oxide (NO) production and NMDA receptor stimulation. Our experiments revealed that icilin (2.5, 5, 7.5 and 10 mg/kg, i.m.) elicits a dose-related hyperthermia that is rapid in onset and of long duration. Pretreating rats with N(G)-nitro-L-arginine methyl ester hydrochloride (L-NAME) (10, 25 and 50 mg/kg, i.p.), a non-selective NO synthase inhibitor, attenuated the hyperthermia associated with icilin (7.5 mg/kg, i.m.). Pretreatment with (-)-6-[phosphonomethyl-1,2,3,4,4a,5,6,7,8,8a-decahydro-isoquinoline-2-carboxylate] (LY 235959) (0.25, 0.5 and 1 mg/kg, i.p.), a selective NMDA receptor antagonist, also attenuated the icilin-evoked hyperthermia. The administration of icilin (5 and 100 microg) into the lateral cerebroventricle of rats did not affect body temperature, thus indicating a peripheral site of action. These results indicate that icilin, a TRPM8/TRPA1 agonist, produces a dose-related hyperthermia in rats which requires both NO production and NMDA receptor activation.     

Nociception and TRP Channels

Noxious thermal, mechanical, or chemical stimuli evoke pain through excitation of the peripheral terminals called nociceptor, and many kinds of ionotropic and metabotropic receptors are involved in this process. Capsaicin receptor TRPV1 is a nociceptor-specific ion channel that serves as the molecular target of capsaicin. TRPV1 can be activated not only by capsaicin but also by noxious heat (with a thermal threshold >43 degrees C) or protons (acidification), all of which are known to cause pain in vivo. Studies using TRPV1-deficient mice have shown that TRPV1 is essential for selective modalities of pain sensation and for thermal hyperalgesia. One mechanism underlying inflammatory pain which is initiated by tissue damage/inflammation and characterized by hypersensitivity is sensitization of TRPV1. In addition to TRPV1, there are five thermosensitive ion channels in mammals, all of which belong to the TRP (transient receptor potential) super family. These include TRPV2, TRPV3, TRPV4, TRPM8 and TRPA1. These channels exhibit distinct thermal activation thresholds (> 52 degrees C for TRPV2, > approximately 34-38 degrees C for TRPV3, > approximately 27-35 degrees C for TRPV4, < approximately 25-28 degrees C for TRPM8 and < 17 degrees C for TRPA1) and are expressed in primary sensory neurons as well as other tissues. Some of the thermosensitive TRP channels are likely to be involved in thermal nociception, since their activation thresholds are within the noxious range of temperatures.     

Cough sensors. II. Transient receptor potential membrane receptors on cough sensors

The transient receptor potential (TRP) family of channels is represented by at least six members in primary sensory neurons. These include the TRP vanilloid subtypes 1 (TRPV1), 2, 3, and 4, the cold and menthol receptor TRPM8, and TRPA1. Much interest has been directed to the study of the TRPV1, because capsaicin has been instrumental in discovering the unique role of a subset of primary sensory neurons in causing nociceptive responses, in activating reflex pathways including cough, and in producing neurogenic inflammation. TRPV1 is now regarded as an integrator of diverse sensory modalities because it undergoes marked plasticity and sensitization through a variety of mechanisms, including activation of G-protein-coupled or tyrosine kinase receptors. Evidence in experimental animals and in patients with airway diseases indicates a marked hypersensitivity to cough induced by TRPV1 agonists. Recent studies with newly developed high-affinity and selective TRPV1 antagonists have revealed that TRPV1 inhibition reduces cough induced by citric acid or antigen challenge.     

Impairment by activation of TRPA1 of gastric epithelial restitution in a wound model using RGM1 cell monolayer

We examined the influence of TRPA1 on the epithelial restitution using a rat gastric epithelial cell line RGM1 monolayer. RGM1 cells were inoculated in 24-well plates cultured for 24 hr, and then starved for 24 hr in a culture medium at 37 °C under 5 % CO₂ in air. After obtaining a confluent RGM1 cell monolayer, a round artificial wound of constant size was induced in the center of the cell monolayer using a pencil-type mixer with a rotating silicon tip. The repair process was monitored by quantitatively measuring the area of the epithelial wound (cell-free area). Immediately after the wound induction, cells at the edge of wound started to form lamellipodia, migrating toward the center of wound, and by so doing the cell-free area was decreased over time. The addition of icilin, the TRPA1 agonist, suppressed the recovery of the epithelial wound in a concentration-dependent manner. Likewise, another TRPA1 agonist, ally isothiocyanate, also significantly inhibited the wound repair. In addition, the recovery of the epithelial wound was potently inhibited when the ambient temperature was lowered to 17 °C, the threshold temperature where TRPA1 is known to be activated. By contrast, the wound healing was not affected by either menthol, the TRPM8 agonist, or capsaicin, the TRPV1 agonist. These results showed for the first time that the activation of TRPA1 inhibited the repair of the epithelial wound in the stomach, probably by the suppression of cell migration, and suggested the involvement of TRPA1 in the mechanism of gastric epithelial restitution. Received 8 August 2006; accepted 7 November 2006     

Anti-pruritic and anti-inflammatory effects of natural verbascoside through selective inhibition of temperature-sensitive Ca~(2+)-permeable TRPV3 channel

OBJECTIVE To investigate the molecular mechanism underlying the action of natural verbascoside used as traditional herbal medicine for anti-itch and antiinflammation therapy. METHODS To confirm the inhibitory effect of verbascoside on transient receptor potential(TRP) channels, we performed the whole-cel patch clamp recordings of transiently transfected h TRPs-HEK293 cells. To examine the inhibitory effect of verbascoside on TRPV3 activation-induced itch, thymic stromal lymphopoietin(TSLP) release and proinflammatory response, we adopted a pretreatment by intradermal injection of verbascoside into the mouse neck 30 min before injection of TRPV3 agonist carvacrol into the same site. We counted the bouts of scratching behavior in mice for 30 min and performed q-PCR and Western blotting assay to measure the expression levels of TSLP, inflammatory factors such as tumor necrosis factor(TNF)-α and interleukin(IL)-6 from lysates of mouse neck skin tissues. RESULTS We found that natural verbascoside selectively inhibits TRPV3 in dose-dependent manner over other TRP channels including TRPV1, TRPV4, TRPA1 and TRPM8 channels. Verbascoside attenuates the acute scratching behavior and the release of pruritic mediator TSLP and inflammatory factors TNF-α and IL-6 induced by TRPV3 agonist carvacrol. CONCLUSION Natural verbascoside specifically inhibits TRPV3 channel and alleviates pruritus and inflammation via the channel inhibition. Our observations not only provide a mechanistic explanation for the action of natural verbascoside as herbal medicine used for anti-itch and anti-inflammation therapy, but also demonstrate that specific inhibition of TRPV3 may represent therapeutic strategy for potential treatment of pruritus or dermatitis.     

TRP channel gating physiology

Transient Receptor Potential (TRP) cation channels participate in several processes of vital importance in cell and organism physiology, and have been demonstrated to participate in the detection of sensory stimuli. The thermo TRP's reviewed: TRPV1 (vanilloid 1), TRPM8 (melastatin 8) and TRPA1 (ankyrin-like 1) are known to integrate different chemical and physical stimuli such as changes in temperature and sensing different irritant or pungent compounds. However, despite the physiological importance of these channels the mechanisms by which they detect incoming stimuli, how the sensing domains are coupled to channel gating and how these processes are connected to specific structural regions in the channel are not fully understood, but valuable information is available. Many sites involved in agonist detection have been characterized and gating models that describe many features of the channel's behavior have been put forward. In this review we will survey some of the key findings concerning the structural and molecular mechanisms of TRPV1, TRPA1 and TRPM8 activation.     

Agents in early development for treatment of bladder dysfunction – promise of drugs acting at TRP channels?

Introduction: In the lower urinary tract (LUT) several members of the TRP superfamily are involved in nociception and mechanosensory transduction. Animal studies have suggested a therapeutic potential of some of these channels, including TRPV1, TRPV4, TRPM8, TRPA1, and TRPM4, for treatment of bladder over- and underactivity and bladder pain disorders, but translation of this information to clinical application has been slow.

Areas covered: An update on and discussion of current information on the potential clinical use of TRP channel agonists/antagonists in the treatment of different types of bladder dysfunction. The electronic databases PubMed and Scopus were used to identify relevant clinical and animal studies.

Expert opinion: The therapeutic effect of TRPV1 channel desensitizing agonists (capsaicin, resiniferatoxin, given intravesically) has been convincingly demonstrated in some forms of bladder overactivity. However, so far, the potential of any of the small-molecule TRP channel blockers developed for non-bladder indications and tested in early human trials for safety has not been explored clinically in LUT dysfunction. The adverse effects of hyperthermia and reduction of noxious heat sensation of the first generation TRPV1 blockers have delayed development. Despite lack of translational information, TRP channels remain interesting targets for future LUT drugs.     

TRP channels as emerging targets for pain therapeutics

Background: The transient receptor potential (TRP) superfamily of ion channels are a large and diverse group that have received increased attention in recent years. The sub-family of thermo-TRPs which are regulated by temperature, among other physical and chemical stimuli, are of particular interest for the development of potential pain therapeutics. Objective/methods: We review the advances in the field in recent years, focusing on a rationale for pain therapy and potential challenges associated with these targets. Results/conclusions: Vanilloid-type TRP 1 (TRPV1) is the most well studied and advanced member of the family, with selective agonists and antagonists already in clinical use or development, respectively. Among other thermo-TRPs (including TRPV2 – 4, Ankyrin type TRP 1 (TRPA1) and melastatin type TRP 8 (TRPM8)), TRPA1 and TRPM8 are emerging as promising novel pain targets.     

TRP channels: potential drug target for neuropathic pain

Neuropathic pain is a debilitating disease which affects central as well as peripheral nervous system. Transient receptor potential (TRP) channels are ligand-gated ion channels that detect physical and chemical stimuli and promote painful sensations via nociceptor activation. TRP channels have physiological role in the mechanisms controlling several physiological responses like temperature and mechanical sensations, response to painful stimuli, taste, and pheromones. TRP channel family involves six different TRPs (TRPV1, TRPV2, TRPV3, TRPV4, TRPM8, and TRPA1) which are expressed in pain sensing neurons and primary afferent nociceptors. They function as transducers for mechanical, chemical, and thermal stimuli into inward currents, an essential first step for provoking pain sensations. TRP ion channels activated by temperature (thermo TRPs) are important molecular players in acute, inflammatory, and chronic pain states. Different degree of heat activates four TRP channels (TRPV1–4), while cold temperature ranging from affable to painful activate two indistinctly related thermo TRP channels (TRPM8 and TRPA1). Targeting primary afferent nociceptive neurons containing TRP channels that play pivotal role in revealing physical stimuli may be an effective target for the development of successful pharmacotherapeutics for clinical pain syndromes. In this review, we highlighted the potential role of various TRP channels in different types of neuropathic pain. We also discussed the pharmacological activity of naturally and synthetically originated TRP channel modulators for pharmacotherapeutics of nociception and neuropathic pain.     

Molecular mechanisms of thermosensation

We feel a wide range of temperatures spanning from cold to heat. Within this range, temperatures over about 43 degrees C and below about 15 degrees C evoke not only a thermal sensation, but also a feeling of pain. In mammals, six thermosensitive ion channels have been reported, all of which belong to the TRP (transient receptor potential) super family. These include TRPV1 (VR1), TRPV2 (VRL-1), TRPV3, TRPV4, TRPM8 (CMR1), and TRPA1 (ANKTM1). These channels exhibit distinct thermal activation thresholds (>43 degrees C for TRPV1, >52 degrees C for TRPV2, >32-39 degrees C for TRPV3, >27-35 degrees C for TRPV4, <25-28 degrees C for TRPM8, and <17 degrees C for TRPA1) and are expressed in primary sensory neurons as well as other tissues. The involvement of TRPV1 in thermal nociception has been demonstrated by multiple methods, including the analysis of TRPV1-deficient mice. Temperature thresholds for activation of TRPV1, TRPV4, and TRPM8 are not fixed but changeable. Reduction of the temperature threshold for TRPV1 activation is thought to be one mechanism of inflammatory pain. Significant advances in thermosensation research have been made in the last several years with the cloning and characterization of thermosensitive TRP channels. With these clones in hand, we can begin to understand thermosensation from a molecular standpoint.