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.

     

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.     

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)。     

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.     

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.     

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.     

Activation of TRPA1 channels by the fatty acid amide hydrolase inhibitor 3'-carbamoylbiphenyl-3-yl cyclohexylcarbamate (URB597)

As a member of the transient receptor potential (TRP) ion channel superfamily, the ligand-gated ion channel TRPA1 has been implicated in nociceptive function and pain states. The endogenous ligands that activate TRPA1 remain unknown. However, various agonists have been identified, including environmental irritants (e.g., acrolein) and ingredients of pungent natural products [e.g., allyl isothiocyanate (ITC), cinnamaldehyde, allicin, and gingerol]. In general, these agents are either highly reactive, nonselective, or not potent or efficacious, significantly limiting their utilities in the study of TRPA1 channel properties and biological functions. In a search for novel TRPA1 agonists, we identified 3'-carbamoylbiphenyl-3-yl cyclohexylcarbamate (URB597), a potent and systemically active inhibitor of fatty acid amide hydrolase (FAAH). This enzyme is responsible for anandamide degradation and therefore has been pursued as an antinociceptive and antiepileptic drug target. Using Ca(2+) influx assays and patch-clamp techniques, we demonstrated that URB597 could activate heterologously expressed human and rat TRPA1 channels, whereas two other FAAH inhibitors (i.e., URB532 and Compound 7) had no effect. When applied to inside-out membrane patches expressing rat TRPA1, URB597 elicited single-channel activities with a unitary conductance of 40 pS. Furthermore, URB597 activated TRPA1 channels endogenously expressed in a population of rat dorsal root ganglion neurons that also responded to ITC. In contrast to its effect on TRPA1, URB597 inhibited TRPM8 and had no effects on TRPV1 or TRPV4. Thus, we conclude that URB597 is a novel agonist of TRPA1 and probably activates the channel through a direct gating mechanism.     

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.     

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.     

Function and pharmacology of TRPM cation channels

The physiological function and cellular role of some members of the TRPM family are poorly understood and still mysterious. Melastatin, the founding member of the TRPM group, is the most prominent example of the mysteries involved in understanding TRP channel function. Melastatin or TRPM1 was first cloned in 1998 and since then it has been suggested that it functions as a tumor suppressor protein in melanocytes. On the other hand, TRPM8 and TRPA1 have been described as cold receptors, TRPM4 and TRPM5 as calcium-activated nonselective cation channels, TRPM6 and TRPM7 as magnesium-permeable and magnesium-modulated cation channels, TRPM2 as an ADP-ribose-activated channel of macrophages, and TRPM3 as a hypo-osmolarity- and sphingosine-activated channel. There are many unsolved questions and many studies have to be performed to understand the overall function of the TRPM family. In addition to electrophysiological recordings and biochemical characterization, the use of compounds modulating TRPM channel function has often been helpful to study TRPM channels in a cellular context. Therefore, the review will summarize the known functions, activation mechanisms, and pharmacological modulations of the TRPM channels.     

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.     

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.     

Characterization of the mouse cold-menthol receptor TRPM8 and vanilloid receptor type-1 VR1 using a fluorometric imaging plate reader (FLIPR) assay

1. TRPM8 (CMR1) is a Ca(2+)-permeable channel, which can be activated by low temperatures, menthol, eucalyptol and icilin. It belongs to the transient receptor potential (TRP) family, and therefore is related to vanilloid receptor type-1 (VR1, TRPV1). We tested whether substances which are structurally related to menthol, or which produce a cooling sensation, could activate TRPM8, and compared the responses of TRPM8 and VR1 to these ligands. 2. The effects of 70 odorants and menthol-related substances on recombinant mouse TRPM8 (mTRPM8), expressed in HEK293 cells, were examined using a FLIPR assay. In all, 10 substances (linalool, geraniol, hydroxycitronellal, WS-3, WS-23, FrescolatMGA, FrescolatML, PMD38, CoolactP and Cooling Agent 10) were found to be agonists. 3. The EC(50) values of the agonists defined their relative potencies: icilin (0.2+/-0.1 microM)>FrescolatML (3.3+/-1.5 microM) > WS-3 (3.7+/-1.7 microM) >(-)menthol (4.1+/-1.3 microM) >frescolatMAG (4.8+/-1.1 microM) > cooling agent 10 (6+/-2.2 microM) >(+)menthol (14.4+/-1.3 microM) > PMD38 (31+/-1.1 microM) > WS-23 (44+/-7.3 microM) > Coolact P (66+/-20 microM) > geraniol (5.9+/-1.6 mM) > linalool (6.7+/-2.0 mM) > eucalyptol (7.7+/-2.0 mM) > hydroxycitronellal (19.6+/-2.2 mM). 4. Known VR1 antagonists (BCTC, thio-BCTC and capsazepine) were also able to block the response of TRPM8 to menthol (IC(50): 0.8+/-1.0, 3.5+/-1.1 and 18+/-1.1 microM, respectively). 5. The Ca(2+) response of hVR1-transfected HEK293 cells to the endogenous VR1 agonist N-arachidonoyl-dopamine was potentiated by low pH. In contrast, menthol- and icilin-activated TRPM8 currents were suppressed by low pH. 6. In conclusion, in the present study, we identified 10 new agonists and three antagonists of TRPM8. We found that, in contrast to VR1, TRPM8 is inhibited rather than potentiated by protons.     

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.     

TRP Channels in Respiratory Pathophysiology: The Role of Oxidative,Chemical Irritant and Temperature Stimuli

There is rapidly growing evidence indicating multiple and important roles of Ca²⁺-permeable cation TRP channels in the airways, both under normal and disease conditions. The aim of this review was to summarize the current knowledge of TRP channels in sensing oxidative, chemical irritant and temperature stimuli by discussing expression and function of several TRP channels in relevant cell types within the respiratory tract, ranging from sensory neurons to airway smooth muscle and epithelial cells. Several of these channels, such as TRPM2, TRPM8, TRPA1 and TRPV1, are discussed in much detail to show that they perform diverse, and often overlapping or contributory, roles in airway hyperreactivity, inflammation, asthma, chronic obstructive pulmonary disease and other respiratory disorders. These include TRPM2 involvement in the disruption of the bronchial epithelial tight junctions during oxidative stress, important roles of TRPA1 and TRPV1 channels in airway inflammation, hyperresponsiveness, chronic cough, and hyperplasia of airway smooth muscles, as well as TRPM8 role in COPD and mucus hypersecretion. Thus, there is increasing evidence that TRP channels not only function as an integral part of the important endogenous protective mechanisms of the respiratory tract capable of detecting and ensuring proper physiological responses to various oxidative, chemical irritant and temperature stimuli, but that altered expression, activation and regulation of these channels may also contribute to the pathogenesis of respiratory diseases.     

Inhibition of TRPM2 cation channels by N-(p-amylcinnamoyl)anthranilic acid

1. TRPM2 is a Ca2+ -permeable nonselective cation channel activated by intracellular ADP-ribose (ADPR) and by hydrogen peroxide (H2O2). We investigated the modulation of TRPM2 activity by N-(p-amylcinnamoyl)anthranilic acid (ACA). ACA has previously been reported to inhibit phospholipase A2 (PLA2). 2. Using patch-clamp and calcium-imaging techniques, we show that extracellular application of 20 microM ACA completely blocked ADPR-induced whole-cell currents and H2O2-induced Ca2+ signals (IC50 = 1.7 microM) in HEK293 cells transfected with human TRPM2. Two other PLA2 inhibitors, p-bromophenacyl bromide (BPB; 100 microM) and arachidonyl trifluoromethyl ketone (20 microM), had no significant effect on ADPR-stimulated TRPM2 activity. 3. Inhibition of TRPM2 whole-cell currents by ACA was voltage independent and accelerated at decreased pH. ACA was ineffective when applied intracellularly. The single-channel conductance was not changed during ACA treatment, suggesting a reduction of TRPM2 open probability by modulating channel gating. 4. ACA (20 microM) also blocked currents through human TRPM8 and TRPC6 expressed in HEK293 cells, while BPB (100 microM) was ineffective. TRPC6-mediated currents (IC50 = 2.3 microM) and TRPM8-induced Ca2+ signals (IC50 = 3.9 microM) were blocked in a concentration-dependent manner. 5. ADPR-induced currents in human U937 cells, endogeneously expressing TRPM2 protein, were fully suppressed by 20 microM ACA. 6. Our data indicate that ACA modulates the activity of different TRP channels independent of PLA2 inhibition. Owing to its high potency and efficacy ACA can serve, in combination with other blockers, as a useful tool for studying the unknown function of TRPM2 in native cells.     

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.     

Rapid and contrasting effects of rosiglitazone on transient receptor potential TRPM3 and TRPC5 channels

The aim of this study was to generate new insight into chemical regulation of transient receptor potential (TRP) channels with relevance to glucose homeostasis and the metabolic syndrome. Human TRP melastatin 2 (TRPM2), TRPM3, and TRP canonical 5 (TRPC5) were conditionally overexpressed in human embryonic kidney 293 cells and studied by using calcium-measurement and patch-clamp techniques. Rosiglitazone and other peroxisome proliferator-activated receptor-γ (PPAR-γ) agonists were investigated. TRPM2 was unaffected by rosiglitazone at concentrations up to 10 μM but was inhibited completely at higher concentrations (IC(50), ～22.5 μM). TRPM3 was more potently inhibited, with effects occurring in a biphasic concentration-dependent manner such that there was approximately 20% inhibition at low concentrations (0.1-1 μM) and full inhibition at higher concentrations (IC(50), 5-10 μM). PPAR-γ antagonism by 2-chloro-5-nitrobenzanilide (GW9662) did not prevent inhibition of TRPM3 by rosiglitazone. TRPC5 was strongly stimulated by rosiglitazone at concentrations of ≥10 μM (EC(50), ～30 μM). Effects on TRPM3 and TRPC5 occurred rapidly and reversibly. Troglitazone and pioglitazone inhibited TRPM3 (IC(50), 12 μM) but lacked effect on TRPC5, suggesting no relevance of PPAR-γ or the thiazolidinedione moiety to rosiglitazone stimulation of TRPC5. A rosiglitazone-related but nonthiazolidinedione PPAR-γ agonist, N-(2-benzoylphenyl)-O-[2-(methyl-2-pyridinylamino)ethyl]-l-tyrosine (GW1929), was a weak stimulator of TRPM3 and TRPC5. The natural PPAR-γ agonist 15-deoxy prostaglandin J(2), had no effect on TRPM3 or TRPC5. The data suggest that rosiglitazone contains chemical moieties that rapidly, strongly, and differentially modulate TRP channels independently of PPAR-γ, potentially contributing to biological consequences of the agent and providing the basis for novel TRP channel pharmacology.