Topical borneol relieves nonhistaminergic pruritus via targeting TRPA1 and TRPM8 channels in peripheral nerve terminals of mice.

Borneol has been used successfully for the treatment of itchy skin in traditional Chinese medicine. However, the antipruritic effect of borneol has rarely been studied, and the mechanism is unclear. Here, we showed that topical application of borneol on skin substantially suppressed pruritogen chloroquine- and compound 48/80-induced itching in mice. The potential targets of borneol, including transient receptor potential cation channel subfamily V member 3 (TRPV3), transient receptor potential cation channel subfamily A member 1 (TRPA1), transient receptor potential cation channel subfamily M member 8 (TRPM8), and gamma-aminobutyric acid type A (GABAA) receptor were pharmacologically inhibited or genetically knocked out one by one in mouse. Itching behavior studies demonstrated that the antipruritic effect of borneol is largely independent of TRPV3 and GABAA receptor, and TRPA1 and TRPM8 channels are responsible for a major portion of the effect of borneol on chloroquine-induced nonhistaminergic itching. Borneol activates TRPM8 and inhibits TRPA1 in sensory neurons of mice. Topical co-application of TRPA1 antagonist and TRPM8 agonist mimicked the effect of borneol on chloroquine-induced itching. Intrathecal injection of a group II metabotropic glutamate receptor antagonist partially attenuated the effect of borneol and completely abolished the effect of TRPM8 agonist on chloroquine-induced itching, suggesting that a spinal glutamatergic mechanism is involved. In contrast, the effect of borneol on compound 48/80-induced histaminergic itching occurs through TRPA1-and TRPM8-independent mechanisms. Our work demonstrates that borneol is an effective topical itch reliever, and TRPA1 inhibition and TRPM8 activation in peripheral nerve terminals account for its antipruritic effect.

Substantial involvement of TRPM7 inhibition in the therapeutic effect of Ophiocordyceps sinensis on pulmonary hypertension.

Ophiocordyceps sinensis (OCS), an entomopathogenic fungus, is known to exert antiproliferative and antitissue remodeling effects. Vascular remodeling and vasoconstriction play critical roles in the development of pulmonary hypertension (PH). The therapeutic potential of OCS for PH was investigated using rodent PH models, and cultured pulmonary artery endothelial and smooth muscle cells (PAECs and PASMCs), with a focus on the involvement of TRPM7. OCS ameliorated the development of PH, right ventricular hypertrophy and dysfunction in the monocrotaline-induced PH rats. The genetic knockout of TRPM7 attenuated the development of PH in mice with monocrotaline pyrrole-induced PH. TRPM7 was associated with medial hypertrophy and the plexiform lesions in rats and humans with PH. OCS suppressed proliferation of PASMCs derived from the PH patients. Ethanol extracts of OCS inhibited TRPM7-like current, TGF-ß2-induced endothelial-mesenchymal transition, IL-6-induced STAT3 phosphorylation, and PDGF-induced Akt phosphorylation in PAECs or PASMCs. These inhibitory effects were recapitulated by either siRNA-mediated TRPM7 knockdown or treatment with TRPM7 antagonist FTY-720. OCS and FTY-720 induced vasorelaxation in the isolated normal human pulmonary artery. As a result, the present study proposes the therapeutic potential of OCS for the treatment of PH. The inhibition of TRPM7 is suggested to underlie the therapeutic effect of OCS.

The ion channel TRPM7 is required for B cell lymphopoiesis.

The transient receptor potential (TRP) family is a large family of widely expressed ion channels that regulate the intracellular concentration of ions and metals and respond to various chemical and physical stimuli. TRP subfamily M member 7 (TRPM7) is unusual in that it contains both an ion channel and a kinase domain. TRPM7 is a divalent cation channel with preference for Ca2+ and Mg2+ It is required for the survival of DT40 cells, a B cell line; however, deletion of TRPM7 in T cells does not impair their development. We found that expression of TRPM7 was required for B cell development in mice. Mice that lacked TRPM7 in B cells failed to generate peripheral B cells because of a developmental block at the pro-B cell stage. The loss of TRPM7 kinase activity alone did not affect the proportion of peripheral mature B cells or the development of B cells in the bone marrow. However, supplementation with a high concentration of extracellular Mg2+ partially rescued the development of TRPM7-deficient B cells in vitro. Thus, our findings identify a critical role for TRPM7 ion channel activity in B cell development.

Activation of Transient Receptor Potential Melastatin Subtype 8 Attenuates Cold-Induced Hypertension Through Ameliorating Vascular Mitochondrial Dysfunction.

BACKGROUND: Environmental cold-induced hypertension is common, but how to treat cold-induced hypertension remains an obstacle. Transient receptor potential melastatin subtype 8 (TRPM8) is a mild cold-sensing nonselective cation channel that is activated by menthol. Little is known about the effect of TRPM8 activation by menthol on mitochondrial Ca2+ homeostasis and the vascular function in cold-induced hypertension. METHODS AND RESULTS: Primary vascular smooth muscle cells from wild-type or Trpm8-/- mice were cultured. In vitro, we confirmed that sarcoplasmic reticulum-resident TRPM8 participated in the regulation of cellular and mitochondrial Ca2+ homeostasis in the vascular smooth muscle cells. TRPM8 activation by menthol antagonized angiotensin II induced mitochondrial respiratory dysfunction and excess reactive oxygen species generation by preserving pyruvate dehydrogenase activity, which hindered reactive oxygen species-triggered Ca2+ influx and the activation of RhoA/Rho kinase pathway. In vivo, long-term noxious cold stimulation dramatically increased vasoconstriction and blood pressure. The activation of TRPM8 by dietary menthol inhibited vascular reactive oxygen species generation, vasoconstriction, and lowered blood pressure through attenuating excessive mitochondrial reactive oxygen species mediated the activation of RhoA/Rho kinase in a TRPM8-dependent manner. These effects of menthol were further validated in angiotensin II-induced hypertensive mice. CONCLUSIONS: Long-term dietary menthol treatment targeting and preserving mitochondrial function may represent a nonpharmaceutical measure for environmental noxious cold-induced hypertension.

Transient Receptor Potential Cation Channel Subfamily M Member 8 channels mediate the anti-inflammatory effects of eucalyptol.

BACKGROUND AND PURPOSE: Eucalyptol (1,8-cineol), the major ingredient in the essential oil of eucalyptus leaves and other medicinal plants, has long been known for its anti-inflammatory properties. Eucalyptol interacts with the TRP cation channels among other targets, but it is unclear which of these mediates its anti-inflammatory effects. EXPERIMENTAL APPROACH: Effects of eucalyptol were compared in wild-type and TRPM8 channel-deficient mice in two different models: footpad inflammation elicited by complete Freund's adjuvant (CFA) and pulmonary inflammation following administration of LPS. Oedema formation, behavioural inflammatory pain responses, leukocyte infiltration, enzyme activities and cytokine and chemokine levels were measured. KEY RESULTS: In the CFA model, eucalyptol strongly attenuated oedema and mechanical allodynia and reduced levels of inflammatory cytokines (IL-1ß, TNF-α and IL-6), effects comparable with those of ibuprofen. In the LPS model of pulmonary inflammation, eucalyptol treatment diminished leukocyte infiltration, myeloperoxidase activity and production of TNF-α, IL-1ß, IFN-γ and IL-6. Genetic deletion of TRPM8 channels abolished the anti-inflammatory effects of eucalyptol in both models. Eucalyptol was at least sixfold more potent on human, than on mouse TRPM8 channels. A metabolite of eucalyptol, 2-hydroxy-1,8-cineol, also activated human TRPM8 channels. CONCLUSION AND IMPLICATIONS: Among the pharmacological targets of eucalyptol, TRPM8 channels were essential for its anti-inflammatory effects in mice. Human TRPM8 channels are more sensitive to eucalyptol than rodent TRPM8 channels explaining the higher potency of eucalyptol in humans. Metabolites of eucalyptol could contribute to its anti-inflammatory effects. The development of more potent and selective TRPM8 agonists may yield novel anti-inflammatory agents.

Reduction in traumatic brain injury-induced oxidative stress, apoptosis, and calcium entry in rat hippocampus by melatonin: Possible involvement of TRPM2 channels.

Melatonin, which is a very effective reactive oxygen species (ROS) scavenger, acts through a direct reaction with free radicals. Ca(2+) entry induced by traumatic brain injury (TBI) has deleterious effects on human hippocampal function. TRPM2 is a Ca(2+) permeable non-selective channel in hippocampal neurons, and its activation of during oxidative stress has been linked to cell death. Despite the importance of oxidative stress in TBI, its role in apoptosis and Ca(2+) entry in TBI is poorly understood. Therefore, we tested the effects of melatonin on apoptosis, oxidative stress, and Ca(2+) entry through the TRPM2 channel in the hippocampal neurons of TBI-induced rats. Thirty-two rats were divided into the following four groups: control, melatonin, TBI, and TBI + melatonin groups. Melatonin (5 mg/kg body weight) was intraperitoneally given to animals in the melatonin group and the TBI + melatonin group after 1 h of brain trauma. Hippocampal neurons were freshly isolated from the four groups, incubated with a nonspecific TRPM2 blocker (2-aminoethyl diphenylborinate, 2-APB), and then stimulated with cumene hydroperoxide. Apoptosis, caspase-3, caspase-9, intracellular ROS production, mitochondrial membrane depolarization and intracellular free Ca(2+) ([Ca(2+)]i) values were high in the TBI group, and low in the TBI + melatonin group. The [Ca(2+)]i concentration was decreased in the four groups by 2-APB. In our TBI experimental model, TRPM2 channels were involved in Ca(2+) entry-induced neuronal death, and the negative modulation of the activity of this channel by melatonin pretreatment may account for the neuroprotective activity of TRPM2 channels against oxidative stress, apoptosis, and Ca(2+) entry.

TRPM8 is the principal mediator of menthol-induced analgesia of acute and inflammatory pain.

Menthol, the cooling natural product of peppermint, is widely used in medicinal preparations for the relief of acute and inflammatory pain in sports injuries, arthritis, and other painful conditions. Menthol induces the sensation of cooling by activating TRPM8, an ion channel in cold-sensitive peripheral sensory neurons. Recent studies identified additional targets of menthol, including the irritant receptor, TRPA1, voltage-gated ion channels and neurotransmitter receptors. It remains unclear which of these targets contribute to menthol-induced analgesia, or to the irritating side effects associated with menthol therapy. Here, we use genetic and pharmacological approaches in mice to probe the role of TRPM8 in analgesia induced by L-menthol, the predominant analgesic menthol isomer in medicinal preparations. L-menthol effectively diminished pain behavior elicited by chemical stimuli (capsaicin, acrolein, acetic acid), noxious heat, and inflammation (complete Freund's adjuvant). Genetic deletion of TRPM8 completely abolished analgesia by L-menthol in all these models, although other analgesics (acetaminophen) remained effective. Loss of L-menthol-induced analgesia was recapitulated in mice treated with a selective TRPM8 inhibitor, AMG2850. Selective activation of TRPM8 with WS-12, a menthol derivative that we characterized as a specific TRPM8 agonist in cultured sensory neurons and in vivo, also induced TRPM8-dependent analgesia of acute and inflammatory pain. L-menthol- and WS-12-induced analgesia was blocked by naloxone, suggesting activation of endogenous opioid-dependent analgesic pathways. Our data show that TRPM8 is the principal mediator of menthol-induced analgesia of acute and inflammatory pain. In contrast to menthol, selective TRPM8 agonists may produce analgesia more effectively, with diminished side effects.

TRPM7 is regulated by halides through its kinase domain.

Transient receptor potential melastatin 7 (TRPM7) is a divalent-selective cation channel fused to an atypical α-kinase. TRPM7 is a key regulator of cell growth and proliferation, processes accompanied by mandatory cell volume changes. Osmolarity-induced cell volume alterations regulate TRPM7 through molecular crowding of solutes that affect channel activity, including magnesium (Mg(2+)), Mg-nucleotides and a further unidentified factor. Here, we assess whether chloride and related halides can act as negative feedback regulators of TRPM7. We find that chloride and bromide inhibit heterologously expressed TRPM7 in synergy with intracellular Mg(2+) ([Mg(2+)]i) and this is facilitated through the ATP-binding site of the channel's kinase domain. The synergistic block of TRPM7 by chloride and Mg(2+) is not reversed during divalent-free or acidic conditions, indicating a change in protein conformation that leads to channel inactivation. Iodide has the strongest inhibitory effect on TRPM7 at physiological [Mg(2+)]i. Iodide also inhibits endogenous TRPM7-like currents as assessed in MCF-7 breast cancer cells, where upregulation of SLC5A5 sodium-iodide symporter enhances iodide uptake and inhibits cell proliferation. These results indicate that chloride could be an important factor in modulating TRPM7 during osmotic stress and implicate TRPM7 as a possible molecular mechanism contributing to the anti-proliferative characteristics of intracellular iodide accumulation in cancer cells.

Dysregulation of renal transient receptor potential melastatin 6/7 but not paracellin-1 in aldosterone-induced hypertension and kidney damage in a model of hereditary hypomagnesemia.

RATIONALE: Hyperaldosteronism, important in hypertension, is associated with electrolyte alterations, including hypomagnesemia, through unknown mechanisms. OBJECTIVE: To test whether aldosterone influences renal Mg(2+) transporters, (transient receptor potential melastatin (TRPM) 6, TRPM7, paracellin-1) leading to hypomagnesemia, hypertension and target organ damage and whether in a background of magnesium deficiency, this is exaggerated. METHODS AND RESULTS: Aldosterone effects in mice selectively bred for high-normal (MgH) or low (MgL) intracellular Mg(2+) were studied. Male MgH and MgL mice received aldosterone (350 µg/kg per day, 3 weeks). SBP was elevated in MgL. Aldosterone increased blood pressure and albuminuria and increased urinary Mg(2+) concentration in MgH and MgL, with greater effects in MgL. Activity of renal TRPM6 and TRPM7 was lower in vehicle-treated MgL than MgH. Aldosterone increased activity of TRPM6 in MgH and inhibited activity in MgL. TRPM7 and paracellin-1 were unaffected by aldosterone. Aldosterone-induced albuminuria in MgL was associated with increased renal fibrosis, increased oxidative stress, activation of mitogen-activated protein kinases and nuclear factor-NF-κB and podocyte injury. Mg(2+) supplementation (0.75% Mg(2+)) in aldosterone-treated MgL normalized plasma Mg(2+), increased TRPM6 activity and ameliorated hypertension and renal injury. Hence, in a model of inherited hypomagnesemia, TRPM6 and TRPM7, but not paracellin-1, are downregulated. Aldosterone further decreased TRPM6 activity in hypomagnesemic mice, a phenomenon associated with hypertension and kidney damage. Such effects were prevented by Mg(2+) supplementation. CONCLUSION: Amplified target organ damage in aldosterone-induced hypertension in hypomagnesemic conditions is associated with dysfunctional Mg(2+)-sensitive renal TRPM6 channels. Novel mechanisms for renal effects of aldosterone and insights into putative beneficial actions of Mg(2+), particularly in hyperaldosteronism, are identified.

TRPM7 regulates gastrulation during vertebrate embryogenesis.

During gastrulation, cells in the dorsal marginal zone polarize, elongate, align and intercalate to establish the physical body axis of the developing embryo. Here we demonstrate that the bifunctional channel-kinase TRPM7 is specifically required for vertebrate gastrulation. TRPM7 is temporally expressed maternally and throughout development, and is spatially enriched in tissues undergoing convergent extension during gastrulation. Functional studies reveal that TRPM7's ion channel, but not its kinase domain, specifically affects cell polarity and convergent extension movements during gastrulation, independent of mesodermal specification. During gastrulation, the non-canonical Wnt pathway via Dishevelled (Dvl) orchestrates the activities of the GTPases Rho and Rac to control convergent extension movements. We find that TRPM7 functions synergistically with non-canonical Wnt signaling to regulate Rac activity. The phenotype caused by depletion of the Ca(2+)- and Mg(2+)-permeant TRPM7 is suppressed by expression of a dominant negative form of Rac, as well as by Mg(2+) supplementation or by expression of the Mg(2+) transporter SLC41A2. Together, these studies demonstrate an essential role for the ion channel TRPM7 and Mg(2+) in Rac-dependent polarized cell movements during vertebrate gastrulation.

TRPM channels mediate zinc homeostasis and cellular growth during Drosophila larval development.

TRPM channels have emerged as key mediators of diverse physiological functions. However, the ionic permeability relevant to physiological function in vivo remains unclear for most members. We report that the single Drosophila TRPM gene (dTRPM) generates a conductance permeable to divalent cations, especially Zn(2+) and in vivo a loss-of-function mutation in dTRPM disrupts intracellular Zn(2+) homeostasis. TRPM deficiency leads to profound reduction in larval growth resulting from a decrease in cell size and associated defects in mitochondrial structure and function. These phenotypes are cell-autonomous and can be recapitulated in wild-type animals by Zn(2+) depletion. Both the cell size and mitochondrial defect can be rescued by extracellular Zn(2+) supplementation. Thus our results implicate TRPM channels in the regulation of cellular Zn(2+) in vivo. We propose that regulation of Zn(2+) homeostasis through dTRPM channels is required to support molecular processes that mediate class I PI3K-regulated cell growth.

TRPM7 ion channels are required for sustained phosphoinositide 3-kinase signaling in lymphocytes.

Lymphocytes lacking the TRPM7 (transient receptor potential cation channel, subfamily M, member 7) dual function ion channel/protein kinase exhibit a unique phenotype: they are unable to proliferate in regular media, but proliferate normally in media supplemented with 10-15 mM extracellular Mg(2+). Here, we have analyzed the molecular mechanisms underlying this phenotype. We find that upon transition from proliferation-supporting Mg(2+)-supplemented media to regular media, TRPM7-deficient cells rapidly downregulate their rate of growth, resulting in a secondary arrest in proliferation. The downregulated growth rate of transitioning cells is associated with a deactivation of signaling downstream from phosphoinositide 3-kinase, and expression of constitutively active p110 phosphoinositide 3-kinase is sufficient to support growth and proliferation of TRPM7-deficient cells in regular media. Together, these observations indicate that TRPM7 channels are required for sustained phosphoinositide 3-kinase-dependent growth signaling and therefore, that TRPM7 is positioned alongside phosphoinositide 3-kinases as a central regulator of lymphocyte growth and proliferation.

Transient receptor potential (TRP) cation channels: rewarding unique proteins.

The discovery of the Transient Receptor Potential (TRP) superfamily of versatile and polymodal cation channels has dramatically extended our molecular understanding of cellular sensors. The main surprising properties are their diversity in ion selectivity and the polymodal mechanisms of activation, which necessarily result in equally diverse cell functions. They are involved in sensory functions, e.g. the perception of temperature, smell, taste, pain, mechanical signals and respond to many natural compounds used in "traditional medicine". TRP channels are main players in Ca2+ signalling, which controls a plethora of events ranging from neurotransmitter release to gene transcription and cell death. They are also involved in homeostatic functions, e.g. epithelial Ca2+ and Mg2+ reabsorption and lysosomal pH regulation. TRP channel dysfunction contributes to certain human diseases. Finally, TRP channels will become important novel pharmacological targets for the treatment of human diseases and for modulation of sensory functions, e.g. the perception of flavor.

Calcium-activated nonselective cation channels in mammalian cardiomyocytes.

A number of calcium-activated nonselective cation (NSC(Ca)) currents recorded from cardiac preparations have been described in the literature. These currents are implicated in membrane depolarization and in the modulation of cardiac activity. The discovery of a novel family of cation channels, the "transient receptor potential" (TRP) protein family, has revived interest in the study of nonselective cation channels. In particular, the TRPM4 protein provides the basis for an NSC(Ca) channel detected in heart preparations. The role of this channel should not be neglected in the description of our understanding of heart activity and the development of arrhythmias induced by calcium waves. This review focuses on the electrophysiologic and regulatory properties of this native NSC(Ca) channel in cardiac preparations compared with those of the TRPM4 protein. Physiologic and pathologic implications of the current carried by this channel are discussed.

Lysosomal exocytosis is impaired in mucolipidosis type IV.

Mucolipidosis type IV (MLIV) is an autosomal recessive disease characterized by severe neurological impairment, ophthalmologic defects, and gastric dysfunction. MLIV cells have a deficiency in the late endosomal/lysosomal (LEL) pathway that results in the buildup of lysosomal inclusions. Using a Xenopus oocyte expression system, we previously showed that mucolipin-1 (MLN1), the protein encoded by the MCOLN1 gene is a Ca2+ -permeable non-selective cation channel that is transiently modulated by elevations in intracellular Ca2+. We further showed that MLN1 is translocated to the plasma membrane during lysosomal exocytosis. In this study we show that lysosomal exocytosis is impaired in fibroblasts from MLIV patients, indicating that MLN1 plays an active role in this process. Further, we show that transfection with wild type MLN1 cDNA rescues exocytosis, suggesting the possibility of treatments based on the restoration of this crucial cellular function.

Differential expression of capsaicin-, menthol-, and mustard oil-sensitive receptors in naive rat geniculate ganglion neurons.

The roles of capsaicin, menthol, and mustard oils and their receptors in geniculate ganglion (GG) neurons still remain to be elucidated. These receptors belong to the transient receptor potential (TRP) family. Capsaicin-, menthol-, and mustard oil-sensitive receptors are TRPV1, TRPM8, and TRPA1, respectively. The present study aimed to investigate the expression of TRPV1, TRPM8, and TRPA1 in naive rat GG neurons. Furthermore, we examined whether these TRP-expressing GG neurons are myelinated A-fiber or unmyelinated C-fiber neurons. Firstly, using reverse transcription-polymerase chain reaction, TRPV1 mRNA and TRPA1 mRNA were distinctly detected in the naive GG. TRPM8 mRNA was faintly detected. Secondly, using in situ hybridization, TRPV1 mRNA- or TRPA1 mRNA-labeled neurons (signal/noise ratio >or= 10) were observed in 15-20% of GG neurons. Few neurons were labeled by TRPM8 mRNA. Thirdly, neurofilament 200 (NF200) protein, a marker of mylinated A-fiber neurons, was detected in 57% of naive GG neurons. Coexpression of TRPV1 mRNA or TRPA1 mRNA with NF200 was detected in 10% of GG neurons. The present study confirmed the expression of the TRP receptors in the naive GG. The possible roles of TRP receptors in naive GG neurons in somatosensory or gustatory function were suggested.

Molecular identification and functional characterization of a temperature-sensitive transient receptor potential channel (TRPM8) from canine.

TRPM8 belongs to the family of transient receptor potential channels and is activated by cooling and cooling agents, such as icilin and menthol. It is expressed in a subset of sensory neurons and is thought to be involved in thermosensation. Here, we report the cloning and functional characterization of canine TRPM8 (cTRPM8). cTRPM8 shares 95.1%, 94.1%, and 93.9% protein sequence identity with human, rat and mouse TRPM8, respectively. Similar to these mammalian orthologs, cTRPM8 was activated by menthol and icilin with strong outward rectification and little cation selectivity. Menthol and icilin also caused calcium-dependent desensitization. Interestingly, cTRPM8 was activated at <17 degrees C, a temperature threshold lower than that reported for the other orthologs. At 22 degrees C, the EC(50) for activation of cTRPM8 expressed in HEK293 cells by icilin and menthol was 0.06 and 4.3 microM determined by Fluorometric Imaging Plate Reader (FLIPR) and 0.4 and 85 microM by patch clamp, respectively. Mustard oil also activated cTRPM8 (FLIPR EC(50) = 490 microM). Menthol activation was more potent at +60 mV than at -60 mV (EC(50) = 53 and 124 microM, respectively, in Xenopus ooctyes). Icilin-, menthol- and mustard oil-induced intracellular Ca(2+) increases were similarly blocked by N-(4-tertiarybutyl-phenyl)-4-(3-chloropyridin-2-yl) tetrahydropyrazine-1(2H)-carboxamide (BCTC) with IC(50) = 2.3, 2.8 and 1.8 microM, respectively. Cooling-activated current was also inhibited by BCTC. Extracellular calcium blocked cTRPM8 in a concentration- and voltage-dependent manner (half maximal blocking [Ca(2+)] = 1.6 mM at -100 mV). These results constitute the first study of cTRPM8 and support the idea that cTRPM8 functions as a transducer of cold stimuli in vivo.