Activation Of Transient Receptor Potential Vanilloid Research Articles

The Antifibrotic and the Anticarcinogenic Activity of Capsaicin in Hot Chili Pepper in Relation to Oral Submucous Fibrosis

A burning sensation on eating spicy foods purportedly supports the role of capsaicin, an active component of chili peppers, in the etiology of oral submucous fibrosis (OSF). Although the mast cell mediators and activated P2X receptors induce a constant burning sensation through an ATP-dependent mechanism, it is the activation of the transient receptor potential vanilloid 1 (TRPV-1) receptor by capsaicin that aggravates it. The molecular basis for the burning pain in OSF is thus attributable to the activation of TRPV1. There is overwhelming evidence that confirms capsaicin has more of a protective role in attenuating fibrosis and is potentially therapeutic in reversing conditions linked to collagen accumulation. The activation of TRPV-1 by capsaicin increases intracellular calcium ([Ca2+]i), upregulates AMP-activated protein kinase (AMPK) and Sirtuin-1 (SIRT-1), to enrich endothelium-dependent vasodilation via endothelial nitric oxide synthase (eNOS). The induction of vasodilation induces antifibrotic effects by alleviating hypoxia. The antifibrotic effects of capsaicin are mediated through the upregulation of antioxidant enzymes, downregulation of inflammatory genes and suppression of new collagen fibril formation. Capsaicin also demonstrates an anticarcinogenic effect by upregulating the cytotoxic T cells and downregulating regulatory T cells through the inhibition of angiogenesis and promotion of apoptosis. Judicious administration of capsaicin with an appropriate delivery mechanism may have therapeutic benefits in reducing pain sensation, rendering antifibrotic effects, and preventing the malignant transformation of OSF. This paper provides an overview of the molecular basis of capsaicin and its therapeutic application as an antifibrotic and anticarcinogenic agent for the treatment of OSF.

Nitric Oxide Inhibits TRPV4‐mediated Hemichannel Opening in Porcine Nonpigmented Ciliary Epithelium

Previous studies showed the presence of connexin 43 (Cx43), connexin 50 (Cx50), and pannexin 1 (Panx1) in the nonpigmented ciliary epithelium (NPE) of the eye, as well as evidence of hemichannel (HC) activity. In the lens, mechanosensitive Transient Receptor Potential Vanilloid 4 (TRPV4)‐mediated calcium entry stimulates hemichannel activity. The aim here is to test for TRPV4 expression in the NPE, determine whether TRPV4 activation causes HC opening, and study the influence of nitric oxide.Immunohistochemistry was used to probe for TRPV4 in porcine ciliary body. Propidium iodide (PI) uptake and ATP release by primary cultured NPE was used to assess HC opening. PI and ATP are large molecules (MW 668 and 507, respectively) that pass through open hemichannels but not the plasma membrane. ATP in the bathing medium was measured by luciferase luminometry and PI uptake by fluorimetry.Abundant expression of TRPV4 was detected in the NPE cell layer of the ciliary body. Cultured NPE cells displayed an increase in PI uptake when exposed to a TRPV4 agonist GSK 1016790A (10 nM) or to hypoosmotic solution (200 mOsm) (Control 4.65 ± 0.24 vs GSK 10.26 ± 0.32 and Hypo 8.26 ± 0.42 fluorescence units per mg protein; n=6, p<0.001). PI uptake also was increased in NPE cells cultured on a flexible membrane and subjected to cyclic stretch for 1‐10 min (10%, 0.5 Hz), the peak response was at at 2 min (Control 1.5 ± 0.03 vs stretch 2.5 ± 0.10; n=6, p<0.001). The increase in PI uptake caused by hypoosmotic solution or by cyclic stretch was abolished by a selective TRPV4 antagonist HC 067047 (10 μM). Hyposmotic solution increased ATP release from 1.12 ± 0.24 to 2.81± 0.27 (n=12, p<0.001) while cyclic stretch increased ATP release from 0.20 ± 0.03 to 0.40 ± 0.03 pmoles/mg protein (n=6, p<0.001). ATP release responses caused by hypoosmotic solution and cyclic stretch were inhibited by HC 067047. Hypoosmotic solution‐induced ATP release was also inhibited by connexin inhibitory peptide, Gap‐27 (200 μM) and nitric oxide (NO) donors, sodium nitroprusside (SNP) and S‐nitroso‐N‐acetylpenicillamine (SNAP).TRPV4 channels are expressed by the NPE. The ability of HC 067047 and Gap‐27 to prevent ATP release and PI uptake points to TRPV4‐dependent opening of connexin HCs in NPE cells. Suppression of ATP release by SNP and SNAP indicates the hemichannel opening mechanism is inhibited by NO.

Inhibition of SGK1 ameliorates ventriculomegaly in a genetic rat model via regulation of TRPV4 in the choroid plexus

Hydrocephalus is a clinical manifestation of cerebrospinal fluid (CSF) accumulation, leading to ventriculomegaly and increased intracranial pressure. Diuretics and other pharmacotherapies have failed to treat hydrocephalus in the human population; therefore, treatment options remain exclusively surgical. The need to develop pharmacotherapies for the treatment of hydrocephalus remains an unmet clinical need and paramount to this need is the study of brain fluid regulation. Previous studies in our laboratory identified the transient receptor potential vanilloid 4 (TRPV4) channel as a potential regulatory hub protein in the choroid plexus responsible, in part, for the regulation of CSF production. Antagonists of the TRPV4 channel were shown to ameliorate hydrocephalus in a genetic rat model. The current studies highlight a novel drug target: serum‐ and glucocorticoid‐induced kinase 1 (SGK1) in the choroid plexus. A specific small molecular weight, membrane permeant inhibitor of SGK1, SI113, has shown promising results in cancer and metabolic research in recent years. In other tissues, SGK1 has been shown to phosphorylate and activate TRPV4 at the membrane, indicating SGK1 as an important upstream regulatory kinase. SI113 blocked TRPV4‐mediated transepithelial ion flux and barrier conductance increases in a cell culture model of human choroid plexus epithelia (HIBCPP line), and ameliorated ventriculomegaly in the genetic rat model. In hydrocephalic animals, there is an increased apical membrane signal in the choroid plexus epithelial cells via immunofluorescent localization of p‐SGK1 and TRPV4, but not of pan‐SGK1. This is not accompanied by a significant increase in either protein or transcript abundance as measured by western blotting or qPCR, respectively. The change in localization is, however, attenuated by treatment with SI113. Animals treated with SI113 show no change in amounts of TRPV4 or total SGK1, but do show significantly reduced levels of p‐SGK1. Thus, phosphorylation of SGK1 appears to be crucial for the activation of TRPV4 which is, in turn, a channel protein that is activated during the development of hydrocephalus. Like Trpv4‐/‐mice, Sgk1‐/‐mice do not show overt phenotypes, indicating pharmacological antagonism of the kinase would have minimal off‐target effects. Overall, these data provide a strong preclinical basis for the use of SGK1 inhibitors in the treatment of hydrocephalus.

Backbone NMR assignments of the extensive human and chicken TRPV4 N-terminal intrinsically disordered regions as important players in ion channel regulation

Transient receptor potential (TRP) channels are important pharmacological targets due to their ability to act as sensory transducers on the organismic and cellular level, as polymodal signal integrators and because of their role in numerous diseases. However, a detailed molecular understanding of the structural dynamics of TRP channels and their integration into larger cellular signalling networks remains challenging, in part due to the systematic absence of highly dynamic regions pivotal for channel regulation from available structures. In human TRP vanilloid 4 (TRPV4), a ubiquitously expressed homotetrameric cation channel involved in temperature, osmo- and mechano-sensation and in a multitude of (patho)physiological processes, the intrinsically disordered N-terminus encompasses 150 amino acids and thus represents > 17% of the entire channel sequence. Its deletion renders the channel significantly less excitable to agonists supporting a crucial role in TRPV4 activation and regulation. For a structural understanding and a comparison of its properties across species, we determined the NMR backbone assignments of the human and chicken TRPV4 N-terminal IDRs.

Activation of TRPV4 Induces Exocytosis and Ferroptosis in Human Melanoma Cells.

TRPV4 (transient receptor potential vanilloid 4), a calcium permeable TRP ion channel, is known to play a key role in endocytosis. However, whether it contributes to exocytosis remains unclear. Here, we report that activation of TRPV4 induced massive exocytosis in both melanoma A375 cell and heterologous expression systems. We show here that, upon application of TRPV4-specific agonists, prominent vesicle priming from endoplasmic reticulum (ER) was observed, followed by morphological changes of mitochondrial crista may lead to cell ferroptosis. We further identified interactions between TRPV4 and folding/vesicle trafficking proteins, which were triggered by calcium entry through activated TRPV4. This interplay, in turn, enhanced TRPV4-mediated activation of folding and vesicle trafficking proteins to promote exocytosis. Our study revealed a signaling mechanism underlying stimulus-triggered exocytosis in melanoma and highlighted the role of cellular sensor TRPV4 ion channel in mediating ferroptosis.

Role of TRPV1 in High Temperature-Induced Mitochondrial Biogenesis in Skeletal Muscle: A Mini Review.

Transient receptor potential vanilloid 1 (TRPV1) is a protein that is susceptible to cell environment temperature. High temperatures of 40–45°C can activate the TRPV1 channel. TRPV1 is highly expressed in skeletal muscle and located on the sarcoplasmic reticulum (SR). Therefore, TRPV1 activated by high-temperature stress releases Ca2+ from the SR to the cytoplasm. Cellular Ca2+ accumulation is a key event that enhances TRPV1 activity by directly binding to the N-terminus and C-terminus. Moreover, Ca2+ is the key messenger involved in regulating mitochondrial biogenesis in skeletal muscle. Long-term activation of TRPV1 may promote mitochondrial biogenesis in skeletal muscle through the Ca2+-CaMKII-p38 MAPK-PGC-1α signaling axis. The discovery of the TRPV1 channel highlights the potential mechanism for high-temperature stress improving muscle mitochondrial biogenesis. The appropriate hot stimulus in thermal environments might be beneficial to the muscular mitochondrial adaptation for aerobic capacity. However, the investigation of TRPV1 on mitochondrial biogenesis is at an early stage. Further investigations need to examine the role of TRPV1 in response to mitochondrial biogenesis in skeletal muscle induced by different thermal environments.

Protective effects of adipose-derived biogenic nanoparticles on the pulmonary microvascular endothelial barrier in mice with ventilator-induced lung injury via the TRPV4/ROCK1 signalling pathway

PurposeThe "obesity paradox" phenomenon occurs in critically ill patients who receive mechanical ventilation. Our previous studies found that the adipose-derived exosomes secreted by obese mice have a protective effect on the pulmonary microvascular endothelial barrier. However, the extraction of exosomes is cumbersome, their yield is low, and their storage is difficult. After further research, we discovered a new type of adipose-derived bioactive material called: lipoaspirate nanoparticles (Lipo-NPs). MethodsLipo-NPs were extracted and identified using a tangential flow filtration system. The Lipo-NPs were used as an intervention in ventilator-induced lung injury (VILI) models in vivo and in vitro to investigate whether they have a protective effect on lung tissue damage (haematoxylin and eosin staining), lung barrier function (lung wet/dry [W/D] weight ratio, protein concentration in bronchoalveolar lavage fluid (BALF), and Vascular endothelial (VE)-expression), as well as their related mechanisms. ResultsIn both in vivo and in vitro studies, Lipo-NPs can attenuate lung injury, reduce lung W/D ratio and protein concentration in BALF, and augment the expression of the adhesion link-protein VE-cadherin, thus playing a protective role in lung barrier function. This protective effect involves the activation of the transient receptor potential vanilloid 4 (TRPV4)/Rho-associated kinase1 (ROCK1) signalling pathway. We further verified the role of this signalling pathway via activation and inhibition of TRPV4 and ROCK1. Moreover, phosphorylation of myosin light chain 2 (MLC2) regulates F-actin and is a target of the ROCK pathway. ConclusionLipo-NPs can enhance the expression of VE-cadherin by inhibiting the TRPV4/ROCK1/pMLC2 signalling pathway in the mechanical ventilation model, thereby exerting a protective effect on the VILI pulmonary microvascular endothelial barrier.

The effects of vanilloid analogues structurally related to capsaicin on the transient receptor potential vanilloid 1 channel

Transient receptor potential vanilloid 1 (TRPV1) is known as a receptor of capsaicin, a spicy ingredient of chili peppers. It is also sensitive to a variety of pungent compounds and is involved in nociception. Here, we focused on the structural characteristics of capsaicin, and investigated whether vanillylmanderic acid (VMA), vanillic acid (VAcid), vanillyl alcohol (VAlc), vanillyl butyl ether (VBE), and vanillin, containing a vanillyl skeleton similar to capsaicin, affected the TRPV1 activities. For detection of TRPV1 activity, intracellular Ca2+ concentration ([Ca2+]i) was measured in HEK 293 cells heterologously expressing mouse TRPV1 (mTRPV1-HEK) and in mouse sensory neurons. Except for vanillin, four vanilloid analogues dose-dependently increased [Ca2+]i in mTRPV1-HEK. The solutions that dissolved VMA, VAcid and vanillin at high concentrations were acidic, whereas those of VAlc and VBE were neutral. Neutralized VAcid evoked [Ca2+]i increases but neutralized VMA did not. Mutation of capsaicin-sensing sites diminished [Ca2+]i responses to VAcid, VAlc and VBE. VAcid, VMA, and vanillin suppressed the activation of TRPV1 induced by capsaicin. VAcid and VMA also inhibited the acid-induced TRPV1 activation. In sensory neurons, VMA diminished TRPV1 activation by capsaicin or acids. The present data indicate that these structural characteristics of chemical compounds on TRPV1 may provide strategies for the development of novel analgesic drugs targeting nociceptive TRPV1.

Vanilloid agonist-mediated activation of TRPV1 channels requires coordinated movement of the S1–S4 bundle rather than a quiescent state

Transient receptor potential vanilloid1 (TRPV1) channel plays an important role in a wide range of physiological and pathological processes, and a comprehensive understanding of TRPV1 gating will create opportunities for therapeutic intervention. Recent incredible advances in cryo-electron microscopy (cryo-EM) have yielded high-resolution structures of all TRPV subtypes (TRPV1–6) and all of them share highly conserved six transmembrane (TM) domains (S1–S6). As revealed by the open structures of TRPV1 in the presence of a bound vanilloid agonist (capsaicin or resiniferatoxin), TM helicesS1 to S4 form a bundle that remains quiescent during channel activation, highlighting differences in the gating mechanism of TRPV1 and voltage-gated ion channels. Here, however, we argue that the structural dynamics rather than quiescence of S1–S4 domains is necessary for capsaicin-mediated activation of TRPV1. Using fluorescent unnatural amino acid (flUAA) incorporation and voltage-clamp fluorometry (VCF) analysis, we directly observed allostery of the S1–S4 bundle upon capsaicin binding. Covalent occupation of VCF-identified sites, single-channel recording, cell apoptosis analysis, and exploration of the role of PSFL828, a novel non-vanilloid agonist we identified, have collectively confirmed the essential role of this coordinated S1–S4 motility in capsaicin-mediated activation of TRPV1. This study concludes that, in contrast to cryo-EM structural studies, vanilloid agonists are also required for S1–S4 movement during TRPV1 activation. Redefining the gating process of vanilloid agonists and the discovery of new non-vanilloid agonists will allow the evaluation of new strategies aimed at the development of TRPV1 modulators.

Dual action of the cannabinoid receptor 1 ligand arachidonyl-2\u2032-chloroethylamide on calcitonin gene-related peptide release

BackgroundBased on the current understanding of the role of neuropeptide signalling in migraine, we explored the therapeutic potential of a specific cannabinoid agonist. The aim of the present study was to examine the effect of the synthetic endocannabinoid (eCB) analogue, arachidonyl-2′-chloroethylamide (ACEA), on calcitonin gene-related peptide (CGRP) release in the dura and trigeminal ganglion (TG), as cannabinoids are known to activate Gi/o-coupled cannabinoid receptors type 1 (CB1), resulting in neuronal inhibition.MethodsThe experiments were performed using the hemi-skull model and dissected TGs from male Sprague-Dawley rats. CGRP release was induced by either 60 mM K+ (for depolarization-induced stimulation) or 100 nM capsaicin (for transient receptor potential vanilloid 1 (TRPV1) -induced stimulation) and measured using an enzyme-linked immunosorbent assay. The analysis of CGRP release data was combined with immunohistochemistry in order to study the cellular localization of CB1, cannabinoid receptor type 2 (CB2), CGRP and receptor activity modifying protein 1 (RAMP1), a subunit of the functional CGRP receptor, in the TG.ResultsCB1 was predominantly expressed in neuronal somas in which colocalization with CGRP was observed. Furthermore, CB1 exhibited colocalization with RAMP1 in neuronal Aδ-fibres but was not clearly expressed in the CGRP-immunoreactive C-fibres. CB2 was mainly expressed in satellite glial cells and did not show substantial colocalization with either CGRP or RAMP1. Without stimulation, 140 nM ACEA per se caused a significant increase in CGRP release in the dura but not TG, compared to vehicle. Furthermore, 140 nM ACEA did not significantly modify neither K+- nor capsaicin-induced CGRP release. However, when the TRPV1 blocker AMG9810 (1 mM) was coapplied with ACEA, K+-induced CGRP release was significantly attenuated in the TG and dura.ConclusionsResults from the present study indicate that ACEA per se does not exhibit antimigraine potential due to its dual agonistic properties, resulting in activation of both CB1 and TRPV1, and thereby inhibition and stimulation of CGRP release, respectively.

Inhibition of Transient Receptor Potential Vanilloid 4 (TRPV4) Mitigates Seizures.

Astrocytes are critical regulators of the immune/inflammatory response in several human central nervous system (CNS) diseases. Emerging evidence suggests that dysfunctional astrocytes are crucial players in seizures. The objective of this study was to investigate the role of transient receptor potential vanilloid 4 (TRPV4) in 4-aminopyridine (4-AP)-induced seizures and the underlying mechanism. We also provide evidence for the role of Yes-associated protein (YAP) in seizures. 4-AP was administered to mice or primary cultured astrocytes. YAP-specific small interfering RNA (siRNA) was administered to primary cultured astrocytes. Mouse brain tissue and surgical specimens from epileptic patient brains were examined, and the results showed that TRPV4 was upregulated, while astrocytes were activated and polarized to the A1 phenotype. The levels of glial fibrillary acidic protein (GFAP), cytokine production, YAP, signal transducer activator of transcription 3 (STAT3), intracellular Ca2+([Ca2+]i) and the third component of complement (C3) were increased in 4-AP-induced mice and astrocytes. Perturbations in the immune microenvironment in the brain were balanced by TRPV4 inhibition or the manipulation of [Ca2+]iin astrocytes. Knocking down YAP with siRNA significantly inhibited 4-AP-induced pathological changes in astrocytes. Our study demonstrated that astrocytic TRPV4 activation promoted neuroinflammation through the TRPV4/Ca2+/YAP/STAT3 signaling pathway in mice with seizures. Astrocyte TRPV4 inhibition attenuated neuroinflammation, reduced neuronal injury, and improved neurobehavioral function. Targeting astrocytic TRPV4 activation may provide a promising therapeutic approach for managing epilepsy.

Potential use for chronic pain: Poly(Ethylene Glycol)-Poly(Lactic-Co-Glycolic Acid) nanoparticles enhance the effects of Cannabis-Based terpenes on calcium influx in TRPV1-Expressing cells

The objective of these in vitro studies was to investigate the impact of the encapsulation of three cannabis-based terpenes, namely β-myrcene (MC), β-caryophyllene (CPh), and nerolidol (NL), on their potential efficacy in pain management. Terpene-encapsulated poly(ethylene glycol)-poly(lactic-co-glycolic acid) nanoparticles (PEG-PLGA NPs) were prepared by an emulsion-solvent evaporation method. The terpene-loaded NPs were examined in HEK293 cells that express the nociceptive transient receptor potential vanilloid-1 (TRPV1), an ion channel involved in pain perception. TRPV1 activation was assessed by monitoring calcium influx kinetics over 1 h in cells pre-treated with the fluorescent indicator Fluo-4. In addition, the fluorescence intensity changes induced by the NPs in living cells were also explored by a fluorescence microscope. Furthermore, the cytotoxicity of the terpene-loaded NPs was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-3,5-diphenyl tetrazolium bromide (MTT) proliferation assay. The terpene-loaded NPs had a diameter in the range of 250–350 nm and a zeta potential of approximately −20 mV. The encapsulation efficiency was 18.5%, 51.3%, and 60.3% for MC, NL, and CPh NPs, respectively. The nano-formulations significantly increased the fluorescence intensity in comparison with free terpenes. Furthermore, combinations of terpene-loaded NPs produced significantly higher calcium responses when compared to combinations of free terpenes. Similar findings were shown by the fluorescence images. In conclusion, the terpene-PLGA NPs can be promising therapeutics for more effective pain management.

Diverse Roles of TRPV4 in Macrophages: A Need for Unbiased Profiling.

Transient receptor potential vanilloid 4 (TRPV4) is a non-selective mechanosensitive ion channel expressed by various macrophage populations. Recent reports have characterized the role of TRPV4 in shaping the activity and phenotype of macrophages to influence the innate immune response to pathogen exposure and inflammation. TRPV4 has been studied extensively in the context of inflammation and inflammatory pain. Although TRPV4 activity has been generally described as pro-inflammatory, emerging evidence suggests a more complex role where this channel may also contribute to anti-inflammatory activities. However, detailed understanding of how TRPV4 may influence the initiation, maintenance, and resolution of inflammatory disease remains limited. This review highlights recent insights into the cellular processes through which TRPV4 contributes to pathological conditions and immune processes, with a focus on macrophage biology. The potential use of high-throughput and omics methods as an unbiased approach for studying the functional outcomes of TRPV4 activation is also discussed.

Activation of TRPV4 stimulates transepithelial K+ secretion in rat epididymal epithelium

The maturation of sperms is dependent on the coordinated interactions between sperm and the unique epididymal luminal milieu, which is characterized by high K+ content. This study investigated the involvement of transient receptor potential vanilloid 4 (TRPV4) in the K+ secretion of epididymal epithelium. The expression level and cellular localization of TRPV4 and Ca2+-activated K+ channels (KCa) were analyzed via RT-PCR, real-time quantitative PCR, western blot and immunofluorescence. The functional role of TRPV4 was investigated using short-circuit current (ISC) and intracellular Ca2+ imaging techniques. We found a predominant expression of TRPV4 in the corpus and cauda epididymal epithelium. Activation of TRPV4 with a selective agonist, GSK1016790A, stimulated a transient decrease in the ISC of the epididymal epithelium. The ISC response was abolished by either the TRPV4 antagonists, HC067047 and RN-1734, or the removal of basolateral K+. Simultaneously, the application of GSK1016790A triggered Ca2+ influx in epididymal epithelial cells. Our data also indicated that the big conductance KCa (BK), small conductance KCa (SK) and intermediate conductance KCa (IK) were all expressed in rat epididymis. Pharmacological studies revealed that BK, but not SK and IK, mediated TRPV4-elicited transepithelial K+ secretion. Finally, we demonstrated that TRPV4 and BK were localized in the epididymal epithelium, which showed an increased expression level from caput to cauda regions of rat epididymis. This study implicates that TRPV4 plays an important role in the formation of high K+ concentration in epididymal intraluminal fluid via promoting transepithelial K+ secretion mediated by BK.

Cannabinoid and vanilloid pathways mediate opposing forms of synaptic plasticity in corticotropin-releasing hormone neurons.

Activity-dependent release of retrograde signaling molecules form micro-feedback loops to regulate synaptic function in neural circuits. Single neurons can release multiple forms of these signaling molecules, including endocannabinoids and endovanilloids, which act via cannabinoid (CB) receptors and transient receptor potential vanilloid 1 (TRPV1) receptors. In hypothalamic corticotrophin-releasing hormone (CRH) neurons, endocannabinoids acting via CB1 receptors have been shown to play an important role in regulating excitability and hence stress hormone secretion. However, the importance of endovanilloid signaling in CRH neurons is currently unclear. Here, we show that, in response to postsynaptic depolarization, CRH neurons release endocannabinoid/endovanilloid molecules that can activate CB1 and TRPV1 receptors. Activation of CB1 receptors suppresses glutamate neurotransmission whereas activation of TRPV1 enhances spontaneous glutamate transmission. However, the excitatory effects of TRPV1 are normally masked by the inhibitory effects of CB1. When the degradation of the endocannabinoid 2-arachidonoylglycerol (2-AG) was inhibited, this revealed tonic activation of CB1 receptors, suggesting tonic endocannabinoid release. However, we found no evidence for tonic activation of TRPV1 receptors under similar conditions. These findings show that activation of CRH neurons can drive the release of signaling molecules that activate parallel endocannabinoid and endovanilloid receptor pathways to mediate opposing forms of synaptic plasticity.

Distribution of transient receptor potential vanilloid-1 channels in gastrointestinal tract of patients with morbid obesity.

BACKGROUNDTransient receptor potential vanilloid-1 (TRPV1), a nonselective cation channel, is activated by capsaicin, a pungent ingredient of hot pepper. Previous studies have suggested a link between obesity and capsaicin-associated pathways, and activation of TRPV1 may provide an alternative approach for obesity treatment. However, data on the TRPV1 distribution in human gastric mucosa are limited, and the degree of TRPV1 distribution in the gastric and duodenal mucosal cells of obese people in comparison with normal-weight individuals is unknown.AIMTo clarify gastric and duodenal mucosal expression of TRPV1 in humans and compare TRPV1 expression in obese and healthy individuals.METHODSForty-six patients with a body mass index (BMI) of > 40 kg/m2 and 20 patients with a BMI between 18-25 kg/m2 were included. Simultaneous biopsies from the fundus, antrum, and duodenum tissues were obtained from subjects between the ages of 18 and 65 who underwent esophagogastroduodenoscopy. Age, sex, history of alcohol and cigarette consumption, and past medical history regarding chronic diseases and medications were accessed from patient charts and were analyzed accordingly. Evaluation with anti-TRPV1 antibody was performed separately according to cell types in the fundus, antrum, and duodenum tissues using an immunoreactivity score. Data were analyzed using SPSS 17.0.RESULTSTRPV1 expression was higher in the stomach than in the duodenum and was predominantly found in parietal and chief cells of the fundus and mucous and foveolar cells of the antrum. Unlike foveolar cells in the antrum, TRPV1 was relatively low in foveolar cells in the fundus (4.92 ± 0.49 vs 0.48 ± 0.16, P < 0.01, Mann-Whitney U test). Additionally, the mucous cells in the duodenum also had low levels of TRPV1 compared to mucous cells in the antrum (1.33 ± 0.31 vs 2.95 ± 0.46, P < 0.01, Mann-Whitney U test). TRPV1 expression levels of different cell types in the fundus, antrum, and duodenum tissues of the morbidly obese group were similar to those of the control group. Staining with TRPV1 in fundus chief cells and antrum and duodenum mucous cells was higher in patients aged ≥ 45 years than in patients < 45 years (3.03 ± 0.42, 4.37 ± 0.76, 2.28 ± 0.55 vs 1.9 ± 0.46, 1.58 ± 0.44, 0.37 ± 0.18, P = 0.03, P < 0.01, P < 0.01, respectively, Mann-Whitney U test). The mean staining levels of TRPV1 in duodenal mucous cells in patients with diabetes and hypertension were higher than those in patients without diabetes and hypertension (diabetes: 2.11 ± 0.67 vs 1.02 ± 0.34, P = 0.04; hypertension: 2.42 ± 0.75 vs 1.02 ± 0.33, P < 0.01 Mann-Whitney U test).CONCLUSIONThe expression of TRPV1 is unchanged in the gastroduodenal mucosa of morbidly obese patients demonstrating that drugs targeting TRPV1 may be effective in these patients.

Cardiac TRPV4 channels.

Transient Receptor Potential Vanilloid 4 (TRPV4) is expressed in numerous cell types within the heart, yet the expression levels, subcellular localization, and functional relevance of TRPV4 in cardiac myocytes is under-appreciated. Recent data indicate a critical role of TRPV4 in both atrial and ventricular myocyte biology, with expression levels and channel function increasing following pathological scenarios including ischemia, myocardial infarction, mechanical stress, and inflammation. Excessive activation of TRPV4 at the cellular level contributes to enhanced Ca2+ entry which predisposes the cardiac myocyte to pro-arrhythmic Ca2+ overload and electrophysiological abnormalities. At the organ level, excessive TRPV4 activity associates with cardiac hypercontractility, cardiac damage, ventricular arrhythmia, and atrial fibrillation. This manuscript chapter describes the emerging literature on TRPV4 in cardiac myocytes in physiology and disease.

TRPV4-induced M\xfcller cell gliosis and TNF-\u03b1 elevation-mediated retinal ganglion cell apoptosis in glaucomatous rats via JAK2/STAT3/NF-\u03baB pathway

BackgroundGlaucoma, the leading cause of irreversible blindness worldwide, is a type of retinal disease characterized by the selective death of retinal ganglion cells (RGCs). However, the pathogenesis of glaucoma has not been fully elucidated. Transient receptor potential vanilloid 4 (TRPV4) is a pressure-sensitive and calcium-permeable cation channel. TRPV4 is widely distributed in the retina and its sustained activation leads to RGC death; indicating that TRPV4 may be a possible target for glaucoma treatment. Here, we investigated the effects of TRPV4 on RGC apoptosis in a rat model of chronic ocular hypertension (COH), then examined the mechanism underlying these effects.MethodsThe COH model was established by injection of micro-magnetic beads into the anterior chamber of adult male rats. The expression levels of TRPV4, glial fibrillary acidic protein, and inflammatory factors were assessed by immunohistochemistry and immunoblotting. RGC apoptosis and visual dysfunction were evaluated by TUNEL assay and photopic negative response. Functional expression of TRPV4 was examined by electrophysiology and calcium imaging. Real-time polymerase chain reaction and immunoblotting were employed to investigate the molecular mechanism underlying the effects of TRPV4 on tumor necrosis factor-α (TNF-α) release.ResultsWe found that TRPV4 played an essential role in glaucoma, such that high levels of TRPV4 expression were associated with elevated intraocular pressure. Furthermore, TRPV4 activation was involved in glaucoma-induced RGC apoptosis and RGC-related reductions in visual function. Mechanistic investigation demonstrated that TRPV4 activation led to enhanced Müller cell gliosis and TNF-α release via the JAK2/STAT3/NF-kB pathway, while TRPV4 inhibition could reverse these effects. Finally, TRPV4 activation could lead to elevated expression of TNF receptor 1 in RGCs, while inhibition of TNF-α could reduce TRPV4-mediated RGC apoptosis.ConclusionsTRPV4 activation induces Müller cell gliosis and TNF-α elevation via the JAK2/STAT3/NF-κB pathway, which may exacerbate RGC apoptosis in glaucoma; these results suggest that TRPV4 can serve as a therapeutic target in glaucoma treatment.

Abstract 13709: TRPV4 Enhances Ca 2+ Transients and Promotes Arrhythmic Ca 2+ Signaling Following Hypoosmotic Stress and Ischemia-Reperfusion

Introduction: Transient Receptor Potential Vanilloid 4 (TRPV4) is an osmotically activated Ca 2+ channel which enhances cardiomyocyte Ca 2+ cycling and may contribute to arrhythmic Ca 2+ signals in hypoosmotic stress and Ischemia-Reperfusion (I/R). Hypothesis: TRPV4 overexpression enhances Ca 2+ transient (CaT) amplitude and arrhythmic Ca 2+ events in left ventricular (LV) cardiomyocytes exposed to hypoosmotic stress and Langendorff hearts following I/R. Methods: Mouse LV cardiomyocytes from TRPV4 Overexpressors (OEX) and non-transgenic controls (CTL) were exposed to hypoosmotic stress (250 mOsm, 30min). CaT amplitude (paced 0.5 Hz) and arrhythmic Ca 2+ events (prolonged kinetics, spontaneous CaT, Ca 2+ waves) were monitored via confocal imaging (fluo-4, 100Hz). I/R (45min global ischemia, 30min reperfusion) experiments were conducted in OEX and CTL Langendorff hearts expressing the GCaMP6f Ca 2+ sensor (OEX-GCaMP, CTL-GCaMP). CaT and arrhythmic Ca 2+ signals (Ca 2+ waves, alternans, Ca 2+ overload) were monitored in the LV myocardium. Experiments were conducted in the absence and presence of TRPV4 inhibition by HC067047 (HC, 1uM). Results: Incidence of arrhythmic Ca 2+ signals during minutes 0-15 of hypoosmotic stress was increased in OEX cardiomyocytes vs CTL (78 vs 20%, P&lt;0.05, N=9, 5) and prevented in OEX by HC (11%, P&lt;0.05, N=9). Arrhythmia score was increased in OEX vs CTL (1.3±0.7 vs. 0.1±0.1, P&lt;0.05, N=9, 5) and decreased in OEX by HC (0.2±0.2, P&lt;0.05, N=9). Following hypoosmotic stress, CaT amplitude was elevated in OEX vs CTL (F/F 0 = 3.6±0.4 vs 2.1±0.4, P&lt;0.05, N=7, 4) and this effect in OEX was prevented by HC (F/F 0 =2.4±0.4, P&lt;0.05, N=8). Langendorff OEX-GCaMP hearts subjected to I/R demonstrated increased systolic Ca 2+ (F/F 0 =1.8±0.09 vs 1.3±0.1, P&lt;0.05, N=5, 6) during minutes 10-30 of reperfusion, which was attenuated by HC (F/F 0 =1.4±0.08, P&lt;0.05, N=4). OEX-GCaMP hearts were more likely to demonstrate arrhythmic Ca 2+ signals during minutes 15-30 of reperfusion than CTL-GCaMP (27/487 vs 0/415 cells, p&lt;0.05 N=5, 6) and this effect in OEX-GCaMP was prevented by HC (0/633 cells, p&lt;0.05, N=4). Conclusions: TRPV4 activation enhances Ca 2+ transients and promotes arrhythmic Ca 2+ signals, and may provide a novel anti-arrhythmic target in I/R.

Effects of in-vitro modulation of TRPV1 activity on immune response of mice bearing metastatic breast carcinoma: Enhanced inflammatory response may hinder therapeutic potentials of TRPV1 agonists

AimsActivation of transient receptor potential vanilloid 1 (TRPV1) ion channels inhibits inflammation, enhance cytotoxic immune response, and may have therapeutic potential in treatment of cancer characterized by increased systemic inflammation. We here determined how activation of TRPV1 alters immune response of tumor-bearing mice. Main methodsThree different metastatic subset of 4 T1 breast carcinoma cells were used to induce tumors in Balb-c mice. Mix leukocyte cultures (MLCs) using spleens and draining lymph nodes were prepared and stimulated with various challenges. Effects TRPV1 agonists including capsaicin, antagonist (AMG9810) and Gambogic Amide (GA), a TrkA agonist that sensitizes TRPV1, on secreted levels of cytokines were determined. Key findingsMLCs of tumor-bearing mice secreted markedly higher levels of IL-6 and lower levels of IFN-γ compared to control mice. We observed differential effects of TRPV1 agonists in control and mice bearing different subset of metastatic cells. TRPV1 increased IFN–γ and IL-17 secretion in control mice while they markedly increased IL-6 secretion and suppressed IFN--γ secretion in tumor-bearing mice. Unexpectedly, AMG9810 acted as an inverse agonist and did not antagonize the effects of TRPV1 agonists. SignificanceOur results demonstrate constitutive activity of TRPV1 in immune cells, suggesting cross activation. To prevent excessive chronic activation of TRPV1 in immune cells in the presence of metastatic breast carcinoma, lower doses of TRPV1 agonist should be considered. Unexpected findings further document that a drug can have multiple intrinsic activities depending on surrounding factors can act on the same receptor as an agonist, antagonist or inverse agonist.