Expression and distribution of three transient receptor potential vanilloid(TRPV) channel proteins in human odontoblast-like cells

Odontoblasts have been suggested to contribute to nociceptive sensation in the tooth via expression of the transient receptor potential (TRP) channels. The TRP channels as a family of nonselective cation permeable channels play an important role in sensory transduction of human. In this study, we examined the expression of transient receptor potential vanilloid-1 (TRPV1), transient receptor potential vanilloid-2 (TRPV2) and transient receptor potential vanilloid-3 (TRPV3) channels in native human odontoblasts (HODs) and long-term cultured human dental pulp cells with odontoblast phenotyoe (LHOPs) obtained from healthy wisdom teeth with the use of immunohistochemistry (IHC), immunofluorescence (IF), quantitative real-time polymerase chain reaction (qRT-PCR),western blotting (WB) and immunoelectron microscopy (IEM) assay. LHOPs samples were made into ultrathin sections, mounted on nickel grids, floated of three TRPV antibodies conjugated with 10 nm colloidal gold particles and observed under IEM at 60,000 magnifications. The relative intracellular distributions of these three channels were analyzed quantitatively on IEM images using a robust sampling, stereological estimation and statistical evaluation method. The results of IHC and IF convinced that TRPV1, TRPV2 and TRPV3 channels were expressed in native HODs and (LHOPs). The result of qRT-PCR and WB confirmed that the gene and protein expression of TRPV1, TRPV2, and TRPV3 channels and TRPV1 mRNA are more abundantly expressed than TRPV2 and TRPV3 in HODs (P?<?0.05). Quantitative analysis of IEM images showed that the relative intracellular distributions of these three channels are similar, and TRPV1, TRPV2 and TRPV3 proteins were preferential labeled in human odontoblast processes, mitochondria, and endoplasmic reticulum. Thus, HODs could play an important role in mediating pulp thermo-sensation due to the expression of these three TRPV channels. The difference of relative intracellular distributions of three channels suggests that special structures such as processes may have an important role to sensing of the outer stimuli first.     

Transient receptor potential ankyrin 1 channel localized to non-neuronal airway cells promotes non-neurogenic inflammation

Background

The transient receptor potential ankyrin 1 (TRPA1) channel, localized to airway sensory nerves, has been proposed to mediate airway inflammation evoked by allergen and cigarette smoke (CS) in rodents, via a neurogenic mechanism. However the limited clinical evidence for the role of neurogenic inflammation in asthma or chronic obstructive pulmonary disease raises an alternative possibility that airway inflammation is promoted by non-neuronal TRPA1.

Methodology/Principal Findings

By using Real-Time PCR and calcium imaging, we found that cultured human airway cells, including fibroblasts, epithelial and smooth muscle cells express functional TRPA1 channels. By using immunohistochemistry, TRPA1 staining was observed in airway epithelial and smooth muscle cells in sections taken from human airways and lung, and from airways and lung of wild-type, but not TRPA1-deficient mice. In cultured human airway epithelial and smooth muscle cells and fibroblasts, acrolein and CS extract evoked IL-8 release, a response selectively reduced by TRPA1 antagonists. Capsaicin, agonist of the transient receptor potential vanilloid 1 (TRPV1), a channel co-expressed with TRPA1 by airway sensory nerves, and acrolein or CS (TRPA1 agonists), or the neuropeptide substance P (SP), which is released from sensory nerve terminals by capsaicin, acrolein or CS), produced neurogenic inflammation in mouse airways. However, only acrolein and CS, but not capsaicin or SP, released the keratinocyte chemoattractant (CXCL-1/KC, IL-8 analogue) in bronchoalveolar lavage (BAL) fluid of wild-type mice. This effect of TRPA1 agonists was attenuated by TRPA1 antagonism or in TRPA1-deficient mice, but not by pharmacological ablation of sensory nerves.

Conclusions

Our results demonstrate that, although either TRPV1 or TRPA1 activation causes airway neurogenic inflammation, solely TRPA1 activation orchestrates an additional inflammatory response which is not neurogenic. This finding suggests that non-neuronal TRPA1 in the airways is functional and potentially capable of contributing to inflammatory airway diseases.     

TRPA1 is functionally co-expressed with TRPV1 in cardiac muscle: Co-localization at z-discs,costameres and intercalated discs

Transient receptor potential channels of the ankyrin subtype-1 (TRPA1) and vanilloid subtype-1 (TRPV1) are structurally related, non-selective cation channels that show a high permeability to calcium. Previous studies indicate that TRP channels play a prominent role in the regulation of cardiovascular dynamics and homeostasis, but also contribute to the pathophysiology of many diseases and disorders within the cardiovascular system. However, no studies to date have identified the functional expression and/or intracellular localization of TRPA1 in primary adult mouse ventricular cardiomyocytes (CMs). Although TRPV1 has been implicated in the regulation of cardiac function, there is a paucity of information regarding functional expression and localization of TRPV1 in adult CMs. Our current studies demonstrate that TRPA1 and TRPV1 ion channels are co-expressed at the protein level in CMs and both channels are expressed throughout the endocardium, myocardium and epicardium. Moreover, immunocytochemical localization demonstrates that both channels predominantly colocalize at the Z-discs, costameres and intercalated discs. Furthermore, specific TRPA1 and TRPV1 agonists elicit dose-dependent, transient rises in intracellular free calcium concentration ([Ca²⁺]_i) that are abolished in CMs obtained from TRPA1^?/? and TRPV1^?/? mice. Similarly, we observed a dose-dependent attenuation of the TRPA1 and TRPV1 agonist-induced increase in [Ca²⁺]_i when WT CMs were pretreated with increasing concentrations of selective TRPA1 or TRPV1 channel antagonists. In summary, these findings demonstrate functional expression and the precise ultrastructural localization of TRPA1 and TRPV1 ion channels in freshly isolated mouse CMs. Crosstalk between TRPA1 and TRPV1 may be important in mediating cellular signaling events in cardiac muscle.     

Plasma membrane stretch activates transient receptor potential vanilloid and ankyrin channels in Merkel cells from hamster buccal mucosa

Merkel cells (MCs) have been proposed to form a part of the MC-neurite complex with sensory neurons. Many transient receptor potential (TRP) channels have been identified in mammals; however, the activation properties of these channels in oral mucosal MCs remain to be clarified. We investigated the biophysical and pharmacological properties of TRP vanilloid (TRPV)-1, TRPV2, TRPV4, TRP ankyrin (TRPA)-1, and TRP melastatin (TRPM)-8 channels, which are sensitive to osmotic and mechanical stimuli by measurement of intracellular free Ca²⁺ concentration ([Ca²⁺]_i) using fura-2. We also analyzed their localization patterns through immunofluorescence. MCs showed immunoreaction for TRPV1, TRPV2, TRPV4, TRPA1, and TRPM8 channels. In the presence of extracellular Ca²⁺, the hypotonic test solution evoked Ca²⁺ influx. The [Ca²⁺]_i increases were inhibited by TRPV1, TRPV2, TRPV4, or TRPA1 channel antagonists, but not by the TRPM8 channel antagonist. Application of TRPV1, TRPV2, TRPV4, TRPA1, or TRPM8 channel selective agonists elicited transient increases in [Ca²⁺]_i only in the presence of extracellular Ca²⁺. The results indicate that membrane stretching in MCs activates TRPV1, TRPV2, TRPV4, and TRPA1 channels, that it may be involved in synaptic transmission to sensory neurons, and that MCs could contribute to the mechanosensory transduction sequence.     

Liquiritin,a novel inhibitor of TRPV1 and TRPA1, protects against LPS-induced acute lung injury

TRPV1 and TRPA1 are cation channels that play key roles in inflammatory signaling pathways. They are co-expressed on airway C-fibers, where they exert synergistic effects on causing inflammation and cough. Licorice, the root of Glycyrrhiza uralensis, has been widely used in China as an anti-inflammatory and anti-coughing herb. To learn if TRPV1 and TRPA1 might be key targets of the anti-inflammatory and antitussive effects of licorice, we examined liquiritin, the main flavonoid compound and active ingredient of licorice, on agonist-evoked TRPV1 and TRPA1 activation. Liquiritin inhibited capsaicin- and allyl isothiocyanate-evoked TRPV1 and TRPA1 whole-cell currents, respectively, with a similar potency and maximal inhibition. In a mouse acute lung injury (ALI) model induced by the bacterial endotoxin lipopolysaccharide, which involves both TRPV1 and TRPA1, an oral gavage of liquiritin prevented tissue damage and suppressed inflammation and the activation of NF-κB signaling pathway in the lung tissue. Liquiritin also suppressed LPS-induced increase in TRPV1 and TRPA1 protein expression in the lung tissue, as well as TRPV1 and TRPA1 mRNA levels in cells contained in mouse bronchoalveolar lavage fluid. In cultured THP-1 monocytes, liguiritin, or TRPV1 and TRPA1 antagonists capsazepine and HC030031, respectively, diminished not only cytokine-induced upregulation of NF-κB function but also TRPV1 and TRPA1 expression at both protein and mRNA levels. We conclude that the anti-inflammatory and antitussive effects of liquiritin are mediated by the dual inhibition of TRPV1 and TRPA1 channels, which are upregulated in nonneuronal cells through the NF-κB pathway during airway inflammation via a positive feedback mechanism.     

Involvement of Rabring7 in EGF receptor degradation as an E3 ligase

Thermosensitive TRP channels display unique thermal responses, suggesting distinct roles mediating sensory transmission of temperature. However, whether relative expression of these channels in dorsal root ganglia (DRG) is altered in nerve injury is unknown. We developed a multiplex ribonuclease protection assay (RPA) to quantify rat TRPV1, TRPV2, TRPV3, TRPV4, TRPA1, and TRPM8 RNA levels in DRG. We used the multiplex RPA to measure thermosensitive TRP channel RNA levels in DRG from RTX-treated rats (300 microg/kg) or rats with unilateral sciatic nerve chronic constriction injury (CCI). TRPV1 and TRPA1 RNA were significantly decreased in DRG from RTX-treated rats, indicating functional colocalization of TRPA1 and TRPV1 in sensory nociceptors. In DRG from CCI rats, TRPA1, TRPV2, and TRPM8 RNA showed slight but significant increases ipsilateral to peripheral nerve injury. Our findings support the hypothesis that increased TRP channel expression in sensory neurons may contribute to mechanical and cold hypersensitivity.     

Intragastric administration of allyl isothiocyanate increases carbohydrate oxidation via TRPV1 but not TRPA1 in mice

The transient receptor potential (TRP) channel family is composed of a wide variety of cation-permeable channels activated polymodally by various stimuli and is implicated in a variety of cellular functions. Recent investigations have revealed that activation of TRP channels is involved not only in nociception and thermosensation but also in thermoregulation and energy metabolism. We investigated the effect of intragastric administration of TRP channel agonists on changes in energy substrate utilization of mice. Intragastric administration of allyl isothiocyanate (AITC; a typical TRPA1 agonist) markedly increased carbohydrate oxidation but did not affect oxygen consumption. To examine whether TRP channels mediate this increase in carbohydrate oxidation, we used TRPA1 and TRPV1 knockout (KO) mice. Intragastric administration of AITC increased carbohydrate oxidation in TRPA1 KO mice but not in TRPV1 KO mice. Furthermore, AITC dose-dependently increased intracellular calcium ion concentration in cells expressing TRPV1. These findings suggest that AITC might activate TRPV1 and that AITC increased carbohydrate oxidation via TRPV1.     

TRPA1 mediates the antinociceptive properties of the constituent of Crocus sativus L., safranal

Safranal, contained in Crocus sativus L., exerts anti‐inflammatory and analgesic effects. However, the underlying mechanisms for such effects are poorly understood. We explored whether safranal targets the transient receptor potential ankyrin 1 (TRPA1) channel, which in nociceptors mediates pain signals. Safranal by binding to specific cysteine/lysine residues, stimulates TRPA1, but not the TRP vanilloid 1 and 4 channels (TRPV1 and TRPV4), evoking calcium responses and currents in human cells and rat and mouse dorsal root ganglion (DRG) neurons. Genetic deletion or pharmacological blockade of TRPA1 attenuated safranal‐evoked release of calcitonin gene‐related peptide (CGRP) from rat and mouse dorsal spinal cord, and acute nociception in mice. Safranal contracted rat urinary bladder isolated strips in a TRPA1‐dependent manner, behaving as a partial agonist. After exposure to safranal the ability of allyl isothiocyanate (TRPA1 agonist), but not that of capsaicin (TRPV1 agonist) or GSK1016790A (TRPV4 agonist), to evoke currents in DRG neurons, contraction of urinary bladder strips and CGRP release from spinal cord slices in rats, and acute nociception in mice underwent desensitization. As previously shown for other herbal extracts, including petasites or parthenolide, safranal might exert analgesic properties by partial agonism and selective desensitization of the TRPA1 channel.     

Role of TRPM2 and TRPV1 cation channels in cellular responses to radiation-induced DNA damage

Background

Radiation exposure causes DNA damage, and DNA repair systems are essential to rescue damaged cells. Although DNA damage or oxidative stress activates transient receptor potential melastatin 2 (TRPM2) and vanilloid 1 (TRPV1) cation channels, it has not been established whether these TRP channels are involved in cellular responses to radiation-induced DNA damage. Here, we investigated the contribution of TRPM2 and TRPV1 channels to γ-irradiation- and UVB-induced DNA damage responses in human lung cancer A549 cells.

Methods

A549 cells were irradiated with γ-rays (2.0 Gy) or UVB (5–10 mJ/cm²). γH2AX foci, ATM activation, 53BP1 accumulation and EGFR expression were evaluated by immunofluorescence staining. Extracellular ATP concentration was measured by luciferin–luciferase assay. Knockdown of TRPM2 and TRPV1 expression was done by siRNA transfection.

Results

γ-Irradiation-induced γH2AX focus formation, ATM activation, 53BP1 accumulation and EGFR nuclear translocation, which are all associated with DNA repair, were suppressed by knockdown of TRPM2 and TRPV1 channels in A549 cells. Release of ATP, which mediates DNA damage response-associated activation of P2Y receptors, was suppressed by pre-treatment with catalase or knockdown of TRPM2 channel, but not TRPV1 channel. Similarly, UVB-induced γH2AX focus formation was suppressed in TRPM2- and TRPV1-knockdown cells, while UVB-induced ATP release was blocked in TRPM2- but not TRPV1-knockdown cells.

Conclusion

Our results suggest that the activation of TRPM2 channel, which mediates ATP release, and TRPV1 channel plays significant roles in the cellular responses to DNA damage induced by γ-irradiation and UVB irradiation.

General significance

Our results provide a new insight into the function of TRP channels from the viewpoint of radiation biology.     

Activation of serotonin receptor 2 by glucosylsphingosine can be enhanced by TRPA1 but not TRPV1: Implication of a novel glucosylsphingosine-mediated itch pathway

Glucosylsphingosine (GS) is an endogenous sphingolipid that specifically accumulates in the skin of patients with atopic dermatitis (AD). Notably, it was recently found that GS can induce itch sensation by activating serotonin receptor 2A and TRPV4 ion channels. However, it is still uncertain whether other molecules are involved in GS-induced itch sensation. Therefore, by using the calcium imaging technique, we investigated whether serotonin receptor 2 – specifically 2A and 2B – can interact with TRPV1 and TRPA1, because these are representative ion channels in the transmission of itch. As a result, it was found that GS did not activate TRPV1 or TRPA1 per se. Moreover, cells expressing both serotonin receptor 2 and TRPV1 did not show any changes in calcium responses. However, enhanced calcium responses were observed in cells expressing serotonin receptor 2 and TRPA1, suggesting a possible interaction between these two molecules. Similar synergistic effects were also observed in cells expressing serotonin receptor 2 and TRPA1, but not TRPV1. Furthermore, a phospholipase C inhibitor (U73122) and a store-operated calcium entry blocker (SKF96365) significantly reduced GS-induced responses in cells expressing both serotonin receptor 2 and TRPA1, but not with pre-treatment with a Gβγ-complex blocker (gallein). Therefore, we propose a putative novel pathway for GS-induced itch sensation, such that serotonin receptor 2 could be coupled to TRPA1 but not TRPV1 in sensory neurons.     

Site-specific contacts enable distinct modes of TRPV1 regulation by the potassium channel Kvβ1 subunit

Transient receptor potential vanilloid 1 (TRPV1) channel is a multimodal receptor that is responsible for nociceptive, thermal, and mechanical sensations. However, which biomolecular partners specifically interact with TRPV1 remains to be elucidated. Here, we used cDNA library screening of genes from mouse dorsal root ganglia combined with patch-clamp electrophysiology to identify the voltage-gated potassium channel auxiliary subunit Kvβ1 physically interacting with TRPV1 channel and regulating its function. The interaction was validated in situ using endogenous dorsal root ganglia neurons, as well as a recombinant expression model in HEK 293T cells. The presence of Kvβ1 enhanced the expression stability of TRPV1 channels on the plasma membrane and the nociceptive current density. Surprisingly, Kvβ1 interaction also shifted the temperature threshold for TRPV1 thermal activation. Using site-specific mapping, we further revealed that Kvβ1 interacted with the membrane-distal domain and membrane-proximal domain of TRPV1 to regulate its membrane expression and temperature-activation threshold, respectively. Our data therefore suggest that Kvβ1 is a key element in the TRPV1 signaling complex and exerts dual regulatory effects in a site-specific manner.     

Functional expression of the transient receptor potential channel TRPA1, a sensor for toxic lung inhalants,in pulmonary epithelial cells

The cation channel TRPA1 functions as a chemosensory protein and is directly activated by a number of noxious inhalants. A pulmonary expression of TRPA1 has been described in sensory nerve endings and its stimulation leads to the acceleration of inflammatory responses in the lung. Whereas the function of TRPA1 in neuronal cells is well defined, only few reports exist suggesting a role in epithelial cells. The aim of the present study was therefore (1) to evaluate the expression of TRPA1 in pulmonary epithelial cell lines, (2) to characterize TRPA1-promoted signaling in these cells, and (3) to study the extra-neuronal expression of this channel in lung tissue sections. Our results revealed that the widely used alveolar type II cell line A549 expresses TRPA1 at the mRNA and protein level. Furthermore, stimulating A549 cells with known TRPA1 activators (i.e., allyl isothiocyanate) led to an increase in intracellular calcium levels, which was sensitive to the TRPA1 blocker ruthenium red. Investigating TRPA1 coupled downstream signaling cascades it was found that TRPA1 activation elicited a stimulation of ERK1/2 whereas other MAP kinases were not affected. Finally, using epithelial as well as neuronal markers in immunohistochemical approaches, a non-neuronal TRPA1 protein expression was detected in distal parts of the porcine lung epithelium, which was also found examining human lung sections. TRPA1-positive staining co-localized with both epithelial and neuronal markers underlining the observed epithelial expression pattern. Our findings of a functional expression of TRPA1 in pulmonary epithelial cells provide causal evidence for a non-neuronal TRPA1-mediated control of inflammatory responses elicited upon TRPA1-mediated registration of toxic inhalants in vivo.     

TRPA1 and TRPV1 contribute to iodine antiseptics‐associated pain and allergy

Iodine antiseptics exhibit superior antimicrobial efficacy and do not cause acquired microbial resistance. However, they are underused in comparison with antibiotics in infection treatments, partly because of their adverse effects such as pain and allergy. The cause of these noxious effects is not fully understood, and no specific molecular targets or mechanisms have been discovered. In this study, we show that iodine antiseptics cause pain and promote allergic contact dermatitis in mouse models, and iodine stimulates a subset of sensory neurons that express TRPA1 and TRPV1 channels. In vivo pharmacological inhibition or genetic ablation of these channels indicates that TRPA1 plays a major role in iodine antiseptics‐induced pain and the adjuvant effect of iodine antiseptics on allergic contact dermatitis and that TRPV1 is also involved. We further demonstrate that iodine activates TRPA1 through a redox mechanism but has no direct effects on TRPV1. Our study improves the understanding of the adverse effects of iodine antiseptics and suggests a means to minimize their side effects through local inhibition of TRPA1 and TRPV1 channels.     

Synthesis and biological evaluation of [6]-gingerol analogues as transient receptor potential channel TRPV1 and TRPA1 modulators

In order to explore the structural determinants for the TRPV1 and TRPA1 agonist properties of gingerols, a series of nineteen analogues (1b-5) of racemic [6]-gingerol (1a) was synthesized and tested on TRPV1 and TRPA1 channels. The exploration of the structure-activity relationships, by modulating the three pharmacophoric regions of [6]-gingerol, led to the identification of some selective TRPV1 agonists/desensitizers of TRPV1 channels (3a, 3f, and 4) and of some full TRPA1 antagonists (2c, 2d, 3b, and 3d).     

The pore properties of human nociceptor channel TRPA1 evaluated in single channel recordings

TRPA channels detect stimuli of different sensory modalities, including a broad spectrum of chemosensory stimuli, noxious stimuli associated with tissue damage and inflammation, mechanical stimuli, and thermal stimuli. Despite a growing understanding of potential modulators, agonists, and antagonists for these channels, the exact mechanisms of channel regulation and activation remain mostly unknown or controversial and widely debated. Relatively little is also known about the basic biophysical parameters of both native and heterologously expressed TRPA channels. Here we use conventional single channel inside-out and outside-out patch recording from the human TRPA1 channel transiently expressed in human embryonic kidney 293T cells to characterize the selectivity of the channel for inorganic mono-/divalent and organic monovalent cations in the presence of allylisothiocyanate (AITC). We show the relative permeability of the hTRPA1 channel to inorganic cations to be:and to organic cations:Na(+)(1.0)≥ dimethylamine (0.99)>trimethylamine (0.7)>tetramethylammonium (0.4)>N-methyl-d-glucamine (0.1). Activation of the hTRPA1 channels by AITC appears to recruit the channels to a conformational state with an increased permeability to large organic cations. The pore of the channels in this state can be characterized as dilated by approximately 1-2.5 ?. These findings provide important insight into the basic fundamental properties and function of TRPA1 channels in general and human TRPA1 channel in particular.     

Effects of activation of skin ion channels TRPM8, TRPV1, and TRPA1 on the immune response. Comparison with effects of cold and heat exposure

The effects of pharmacological stimulation of skin ion channels TRPA1, TRPM8, TRPV1 on the immune response are presented. These effects are compared with the effects of different types of temperature exposures - skin cooling, deep cooling, and deep heating. This analysis allows us to clear the differences in the influence on the immune response of thermosensitive ion channels localized in the skin; (2) whether the changes in the immune response under temperature exposures are due to these thermosensitive ion channels. Experiments were performed on Wistar rats. For stimulation of TRPM8 ion channel, an application to the skin of 1% menthol was used, for TRPA1 - 0.04% allylisotiocianate, and for TRPV1 - capsaicin in a concentration of 0.001.The antigen binding in the spleen was two-times stimulated by activation of the cold-sensitive ion channel TRPM8 and much weaker by activation of warm-sensitive TRPV1 (by 15%), and another cold-sensitive ion channel TRPA1 (by 40%). Only the stimulation of TRPA1 significantly (by 140%) increased antibody formation in the spleen, while TRPM8 had practically no effect on this process, and activation of TRPV1 significantly (by 60%) inhibited antibody formation. Stimulation of the TRPM8 ion channel significantly (by 60%) reduced the level of IgG in the blood, which is believed to control of infectious diseases.The obtained results show that pharmacological activation of the skin TRPA1, TRPM8, TRPV1 ion channels can differently affect the immune system. At the epicenter of changes there were the antigen binding and antibody formation in the spleen, as well as the level of IgG in the blood. Exactly stimulation of the TRPM8 ion channel determines the changes in the immune response when only the skin is cooling, while at deep body heating, the changes in the immune response are mostly determined by the activation of the skin TRPV1 ion channel.     

Transient Receptor Potential Vanilloid-1 (TRPV1) and Ankyrin-1 (TRPA1) Participate in Visceral Hyperalgesia in Chronic Water Avoidance Stress Rat Model

Stressfull life events have powerful influences on visceral perception of certain IBS patients. In the present study, we aimed to examine the involvement of TRPV1 and TRPA1 in the stress-induced visceral hyperalgesia. Rats were exposed to 1-h water avoidance stress (WAS) daily for 10 consecutive days. The abdominal withdrawal reflex (AWR) to colorectal distension was assessed at the end of the 10-day period. Western-blotting analysis was applied to investigate the alterations of TRPV1 and TRPA1 in the colonic afferent dorsal root ganglia (DRG). Compared with control rats, the WAS-treated rats demonstrated a significant increase in the AWR with the pressure ≥40 mm Hg (P < 0.05). Meanwhile, in the WAS-treated rats, western-blotting analysis showed significant upregulation of TRPV1 and TRPA1 in the colonic afferent DRG. The results indicate that WAS could induce the upregulation of TRPV1 and TRPA1 in the colonic afferent DRG, and both receptors may be candidate molecules involved in the stress-induced visceral hyperalgesia in rats.     

Calcium regulation by temperature-sensitive transient receptor potential channels in human uveal melanoma cells

Uveal melanoma (UM) is both the most common and fatal intraocular cancer among adults worldwide. As with all types of neoplasia, changes in Ca²⁺ channel regulation can contribute to the onset and progression of this pathological condition. Transient receptor potential channels (TRPs) and cannabinoid receptor type 1 (CB1) are two different types of Ca²⁺ permeation pathways that can be dysregulated during neoplasia. We determined in malignant human UM and healthy uvea and four different UM cell lines whether there is gene and functional expression of TRP subtypes and CB1 since they could serve as drug targets to either prevent or inhibit initiation and progression of UM. RT-PCR, Ca²⁺ transients, immunohistochemistry and planar patch-clamp analysis probed for their gene expression and functional activity, respectively. In UM cells, TRPV1 and TRPM8 gene expression was identified. Capsaicin (CAP), menthol or icilin induced Ca²⁺ transients as well as changes in ion current behavior characteristic of TRPV1 and TRPM8 expression. Such effects were blocked with either La³⁺, capsazepine (CPZ) or BCTC. TRPA1 and CB1 are highly expressed in human uvea, but TRPA1 is not expressed in all UM cell lines. In UM cells, the CB1 agonist, WIN 55,212-2, induced Ca²⁺ transients, which were suppressed by La³⁺ and CPZ whereas CAP-induced Ca²⁺ transients could also be suppressed by CB1 activation. Identification of functional TRPV1, TRPM8, TRPA1 and CB1 expression in these tissues may provide novel drug targets for treatment of this aggressive neoplastic disease.