Structural snapshots of TRPV1 reveal mechanism of polymodal functionality.

Many transient receptor potential (TRP) channels respond to diverse stimuli and conditionally conduct small and large cations. Such functional plasticity is presumably enabled by a uniquely dynamic ion selectivity filter that is regulated by physiological agents. What is currently missing is a "photo series" of intermediate structural states that directly address this hypothesis and reveal specific mechanisms behind such dynamic channel regulation. Here, we exploit cryoelectron microscopy (cryo-EM) to visualize conformational transitions of the capsaicin receptor, TRPV1, as a model to understand how dynamic transitions of the selectivity filter in response to algogenic agents, including protons, vanilloid agonists, and peptide toxins, permit permeation by small and large organic cations. These structures also reveal mechanisms governing ligand binding substates, as well as allosteric coupling between key sites that are proximal to the selectivity filter and cytoplasmic gate. These insights suggest a general framework for understanding how TRP channels function as polymodal signal integrators.

TRPV4 and chloride channels mediate volume sensing in trabecular meshwork cells.

Aqueous humor drainage from the anterior eye determines intraocular pressure (IOP) under homeostatic and pathological conditions. Swelling of the trabecular meshwork (TM) alters its flow resistance but the mechanisms that sense and transduce osmotic gradients remain poorly understood. We investigated TM osmotransduction and its role in calcium and chloride homeostasis using molecular analyses, optical imaging, and electrophysiology. Anisosmotic conditions elicited proportional changes in TM cell volume, with swelling, but not shrinking, evoking elevations in intracellular calcium concentration [Ca2+]TM. Hypotonicity-evoked calcium signals were sensitive to HC067047, a selective blocker of TRPV4 channels, whereas the agonist GSK1016790A promoted swelling under isotonic conditions. TRPV4 inhibition partially suppressed hypotonicity-induced volume increases and reduced the magnitude of the swelling-induced membrane current, with a substantial fraction of the swelling-evoked current abrogated by Cl- channel antagonists 4,4'-diisothiocyanato-2,2'-stilbenedisulfonic acid (DIDS) and niflumic acid. The transcriptome of volume-sensing chloride channel candidates in primary human was dominated by ANO6 transcripts, with moderate expression of ANO3, ANO7, and ANO10 transcripts and low expression of LTTRC genes that encode constituents of the volume-activated anion channel. Imposition of 190 mosM but not 285 mosM hypotonic gradients increased conventional outflow in mouse eyes. TRPV4-mediated cation influx thus works with Cl- efflux to sense and respond to osmotic stress, potentially contributing to pathological swelling, calcium overload, and intracellular signaling that could exacerbate functional disturbances in inflammatory disease and glaucoma.NEW & NOTEWORTHY Intraocular pressure is dynamically regulated by the flow of aqueous humor through paracellular passages within the trabecular meshwork (TM). This study shows hypotonic gradients that expand the TM cell volume and reduce the outflow facility in mouse eyes. The swelling-induced current consists of TRPV4 and chloride components, with TRPV4 as a driver of swelling-induced calcium signaling. TRPV4 inhibition reduced swelling, suggesting a novel treatment for trabeculitis and glaucoma.

Rapid-Onset, Short-Duration Induction of Colorectal Contractions in Anesthetized, Adult, Male Rats.

Substantial clinical and preclinical evidence indicates that transient receptor potential vanilloid 1 (TRPV1) receptors are expressed on terminals of colorectal chemoreceptors and mechanoreceptors and are involved in various rectal hypersensitivity disorders with common features of colorectal overactivity. These stimulatory properties of TRPV1 receptors on colorectal function suggested that brief stimulation of TRPV1 might provide a means of pharmacologically activating the colorectum to induce defecation in patients with an "unresponsive" colorectum. The current studies explored the basic features of TRPV1 receptor-induced contractions of the colorectum in anesthetized rats with and without acute spinal cord injury (aSCI). Cumulative concentration-response curves to intrarectal (IR) capsaicin (CAP) solutions (0.003%-3.0%) were performed in anesthetized aSCI and spinal intact rats. CAP produced an "inverted U," cumulative concentration-response curve with a threshold for inducing colorectal contractions at 0.01% and a peak response at 0.1% and slight decreases in responses up to 3%. Decreases in responses with concentrations >0.1% are due to a rapid desensitization (i.e., ≤30 minutes) of TRPV1 receptors to each successive dose. Desensitization appeared fully recovered within 24 hours in spinal intact rats. Colorectal contractions were completely blocked by atropine, indicating a reflexogenic activation of parasympathetic neurons, and responses were completely unaffected by a neurokinin 2 receptor antagonist, indicating that release of neurokinin A from afferent terminals and subsequent direct contractions of the smooth muscle was not involved. IR administration of three other TRPV1 receptor agonists produced similar results as CAP. SIGNIFICANCE STATEMENT: Individuals with spinal cord injury often lose control of defecation. Time-consuming bowel programs using digital stimulation of the rectum are used to empty the bowel. This study shows that intrarectal administration of the transient receptor potential vanilloid 1 (TRPV1) receptor agonist, capsaicin, can induce rapid-onset, short-duration colorectal contractions capable of inducing defecation in spinal cord injured and intact rats. Therefore, TRPV1 agonists show promise as potential therapeutics to induce defecation in individuals with neurogenic bowel.

Neural and behavioral control in Caenorhabditis elegans by a yellow-light-activatable caged compound.

Caenorhabditis elegans is used as a model system to understand the neural basis of behavior, but application of caged compounds to manipulate and monitor the neural activity is hampered by the innate photophobic response of the nematode to short-wavelength light or by the low temporal resolution of photocontrol. Here, we develop boron dipyrromethene (BODIPY)-derived caged compounds that release bioactive phenol derivatives upon illumination in the yellow wavelength range. We show that activation of the transient receptor potential vanilloid 1 (TRPV1) cation channel by spatially targeted optical uncaging of the TRPV1 agonist N-vanillylnonanamide at 580 nm modulates neural activity. Further, neuronal activation by illumination-induced uncaging enables optical control of the behavior of freely moving C. elegans without inducing a photophobic response and without crosstalk between uncaging and simultaneous fluorescence monitoring of neural activity.

Simulated microgravity-induced oxidative stress and loss of osteogenic potential of osteoblasts can be prevented by protection of primary cilia.

Oxidative stress has been considered to be closely related to spaceflight-induced bone loss; however, mechanism is elusive and there are no effective countermeasures. Using cultured rat calvarial osteoblasts exposed to microgravity simulated by a random positioning machine, this study addressed the hypotheses that microgravity-induced shortening of primary cilia leads to oxidative stress and that primary cilium protection prevents oxidative stress and osteogenesis loss. Microgravity was found to induce oxidative stress (as represented by increased levels of reactive oxygen species (ROS) and malondialdehyde production, and decreased activities of antioxidant enzymes), which was perfectly replicated in osteoblasts growing in NG with abrogated primary cilia (created by transfection of an interfering RNA), suggesting the possibility that shortening of primary cilia leads to oxidative stress. Oxidative stress was accompanied by mitochondrial dysfunction (represented by increased mitochondrial ROS and decreased mitochondrial membrane potential) and intracellular Ca2+ overload, and the latter was found to be caused by increased activity of Ca2+ channel transient receptor potential vanilloid 4 (TRPV4), as also evidenced by TRPV4 agonist GSK1016790A-elicited Ca2+ influx. Supplementation of HC-067047, a specific antagonist of TRPV4, attenuated microgravity-induced mitochondrial dysfunction, oxidative stress, and osteogenesis loss. Although TRPV4 was found localized in primary cilia and expressed at low levels in NG, microgravity-induced shortening of primary cilia led to increased TRPV4 levels and Ca2+ influx. When primary cilia were protected by miR-129-3p overexpression or supplementation with a natural flavonoid moslosooflavone, microgravity-induced increased TRPV4 expression, mitochondrial dysfunction, oxidative stress, and osteogenesis loss were all prevented. Our data revealed a new mechanism that primary cilia function as a controller for TRPV4 expression. Microgravity-induced injury on primary cilia leads to increased expression and overactive channel of TRPV4, causing intracellular Ca2+ overload and oxidative stress, and primary cilium protection could be an effective countermeasure against microgravity-induced oxidative stress and loss of osteogenic potential of osteoblasts.

Genetic and functional diversification of chemosensory pathway receptors in mosquito-borne filarial nematodes.

Lymphatic filariasis (LF) afflicts over 60 million people worldwide and leads to severe pathological outcomes in chronic cases. The nematode parasites (Nematoda: Filarioidea) that cause LF require both arthropod (mosquito) intermediate hosts and mammalian definitive hosts for their propagation. The invasion and migration of filarial worms through host tissues are complex and critical to survival, yet little is known about the receptors and signaling pathways that mediate directed migration in these medically important species. In order to better understand the role of chemosensory signaling in filarial worm taxis, we employ comparative genomics, transcriptomics, reverse genetics, and chemical approaches to identify putative chemosensory receptor proteins and perturb chemotaxis phenotypes in filarial worms. We find that chemoreceptor family size is correlated with the presence of environmental (extrahost) stages in nematode life cycles, and that filarial worms contain compact and highly diverged chemoreceptor complements and lineage-specific ion channels that are predicted to operate downstream of chemoreceptor activation. In Brugia malayi, an etiological agent of LF, chemoreceptor expression patterns correspond to distinct parasite migration events across the life cycle. To interrogate the role of chemosensation in the migration of larval worms, arthropod and mammalian infectious stage Brugia parasites were incubated in nicotinamide, an agonist of the nematode transient receptor potential (TRP) channel OSM-9. Exposure of microfilariae to nicotinamide alters intramosquito migration, and exposure of L3s reduces chemotaxis toward host-associated cues in vitro. Nicotinamide also potently modulates thermosensory responses in L3s, suggesting a polymodal sensory role for Brugia osm-9. Reverse genetic studies implicate both Brugia osm-9 and the cyclic nucleotide-gated (CNG) channel subunit tax-4 in larval chemotaxis toward host serum, and these ion channel subunits partially rescue sensory defects in Caenorhabditis elegans osm-9 and tax-4 knock-out strains. Together, these data reveal genetic and functional diversification of chemosensory signaling proteins in filarial worms and encourage a more thorough investigation of clade- and parasite-specific facets of nematode sensory receptor biology.

The role of topical capsaicin gel in pain management during microfocused ultrasound treatment for neck laxity.

BACKGROUND: The transient receptor potential vanilloid 1 (TRPV1) provides a heat and pain sensation (nociception). Capsaicin, a TRPV1 agonist, has been shown to induce a refractory period in the nerve terminal expressing TRPV1 and create long-term nerve terminal defunctionalization. OBJECTIVE: To evaluate the efficacy of capsaicin for pain reduction during microfocused ultrasound with visualization (MFU-V) treatment. METHODS AND MATERIALS: A randomized, split-side study including 24 subjects was conducted. A combined 0.025% capsaicin gel and topical anesthetic were randomly applied on one side of the neck, and a topical anesthetic monotherapy was applied on the contralateral side for 30 min before MFU-V treatment. Pain score (visual analog scale, 0-10) was evaluated at T1 (before MFU-V), T2a (after the 4.5-mm transducer treatment), T2b (after the 3.0-mm transducer treatment), and T3 (after the entire treatment). Side effects were recorded. RESULTS: Mean pain scores at T2a for combined and single regimens were 5.19 (±2.26) and 6.91 (±1.72), respectively (p < 0.001). The capsaicin-treated side had a lower pain score at T2b and T3 (p < 0.001). Redness was longer on the capsaicin-treated side (112.67 vs. 10.68 min, p < 0.001). No other adverse events including contact dermatitis were reported. CONCLUSION: A single application of a combined 0.025% capsaicin gel with topical anesthesia produces a significantly lesser pain score during the MFU-V treatment. Defunctionalization of TRPV1 may explain the alleviation of painful sensations caused by heat from MFU-V.

Endocannabinoid activation of the TRPV1 ion channel is distinct from activation by capsaicin.

Transient receptor potential vanilloid 1 (TRPV1) ion channel serves as the detector for noxious temperature above 42 °C, pungent chemicals like capsaicin, and acidic extracellular pH. This channel has also been shown to function as an ionotropic cannabinoid receptor. Despite the solving of high-resolution three-dimensional structures of TRPV1, how endocannabinoids such as anandamide and N-arachidonoyl dopamine bind to and activate this channel remains largely unknown. Here we employed a combination of patch-clamp recording, site-directed mutagenesis, and molecular docking techniques to investigate how the endocannabinoids structurally bind to and open the TRPV1 ion channel. We found that these endocannabinoid ligands bind to the vanilloid-binding pocket of TRPV1 in the "tail-up, head-down" configuration, similar to capsaicin; however, there is a unique interaction with TRPV1 Y512 residue critical for endocannabinoid activation of TRPV1 channels. These data suggest that a differential structural mechanism is involved in TRPV1 activation by endocannabinoids compared with the classic agonist capsaicin.

Cannabidiol Enhances Microglial Beta-Amyloid Peptide Phagocytosis and Clearance via Vanilloid Family Type 2 Channel Activation.

Alzheimer's disease (AD) is associated with the accumulation and aggregation of amyloid in the brain. The cation channel TRPV2 may mediate the pathological changes in mild cognitive impairment. A high-affinity agonist of TRPV2 named cannabidiol is one of the candidate drugs for AD. However, the molecular mechanism of cannabidiol via TRPV2 in AD remains unknown. The present study investigated whether cannabidiol enhances the phagocytosis and clearance of microglial Aß via the TRPV2 channel. We used a human dataset, mouse primary neuron and microglia cultures, and AD model mice to evaluate TRPV2 expression and the ability of microglial amyloid-ß phagocytosis in vivo and in vitro. The results revealed that TRPV2 expression was reduced in the cortex and hippocampus of AD model mice and AD patients. Cannabidiol enhanced microglial amyloid-ß phagocytosis through TRPV2 activation, which increased the mRNA expression of the phagocytosis-related receptors, but knockdown of TRPV2 or Trem2 rescued the expression. TRPV2-mediated effects were also dependent on PDK1/Akt signaling, a pathway in which autophagy was indispensable. Furthermore, cannabidiol treatment successfully attenuated neuroinflammation while simultaneously improving mitochondrial function and ATP production via TRPV2 activation. Therefore, TRPV2 is proposed as a potential therapeutic target in AD, while CBD is a promising drug candidate for AD.

Identification of a Partial and Selective TRPV1 Agonist CPIPC for Alleviation of Inflammatory Pain.

Transient receptor potential vanilloid 1 (TRPV1) is a non-selective cation channel, predominantly expressed in a subset of peripheral sensory neurons for pain signaling. Topical application of agonist capsaicin for desensitizing TRPV1 currents has been approved for relief of chronic pain. However, the potent TRPV1 capsaicin is not ingestible and even topical capsaicin causes common side effects such as skin irritation, swelling, erythema and pruritus, suggesting that a mild TRPV1 agonist might be helpful for reducing side effects while reliving pain. In this study, we reported on a partial and selective TRPV1 agonist 4-(5-chloropyridin-2-yl)-N-(1H-indazol-6-yl)piperazine-1-carboxamide named CPIPC that was modified based on targeting the residue Arg557, important for conversion between the channel antagonism and agonism. Whole-cell patch clamp recordings indicated a concentration-dependent activation of TRPV1 currents by CPIPC with an EC50 of 1.56 ± 0.13 µM. The maximum efficacy of CPIPC (30 µM) was about 60% of saturated capsaicin (10 µM). Repetitive additions of CPIPC caused TRPV1 current desensitization in both TRPV1-expressing HEK293 cells and dorsal root ganglion (DRG) sensory neurons. Oral administration of CPIPC dose-dependently alleviated inflammatory pain in mice. Further site-directed mutagenesis combined with molecular docking revealed that residue Arg557 is critical for TRPV1 activation by CPIPC. Taken together, we identified a novel partial and selective TRPV1 agonist CPIPC that exhibits antinociceptive activity in mice.

High-Throughput Screening of Transient Receptor Potential Channel 1 Ligands in the Light of the Bioluminescence Resonance Energy Transfer Technique.

Ion channels are attractive drug targets for many therapeutic applications. However, high-throughput screening (HTS) of drug candidates is difficult and remains very expensive. We thus assessed the suitability of the bioluminescence resonance energy transfer (BRET) technique as a new HTS method for ion-channel studies by taking advantage of our recently characterized intra- and intermolecular BRET probes targeting the transient receptor potential vanilloid type 1 (TRPV1) ion channel. These BRET probes monitor conformational changes during TRPV1 gating and subsequent coupling with calmodulin, two molecular events that are intractable using reference techniques such as automated calcium assay (ACA) and automated patch-clamp (APC). We screened the small-sized Prestwick chemical library, encompassing 1200 compounds with high structural diversity, using either intra- and intermolecular BRET probes or ACA. Secondary screening of the detected hits was done using APC. Multiparametric analysis of our results shed light on the capability of calmodulin inhibitors included in the Prestwick library to inhibit TRPV1 activation by capsaicin. BRET was the lead technique for this identification process. Finally, we present data exemplifying the use of intramolecular BRET probes to study other transient receptor potential (TRP) channels and non-TRPs ion channels. Knowing the ease of use of BRET biosensors and the low cost of the BRET technique, these assays may advantageously be included for extending ion-channel drug screening. SIGNIFICANCE STATEMENT: This study screened a chemical library against TRPV1 ion channel using bioluminescence resonance energy transfer (BRET) molecular probes and compared the results with the ones obtained using reference techniques such as automated calcium assay and automated patch-clamp. Multiparametric analysis of our results shed light on the capability of calmodulin antagonists to inhibit chemical activation of TRPV1 and indicates that BRET probes may advantageously be included in ion channel drug screening campaigns.

Release of ATP by TRPV4 activation is dependent upon the expression of AQP2 in renal cells.

Increasing evidence indicates that aquaporins (AQPs) exert an influence in cell signaling by the interplay with the transient receptor potential vanilloid 4 (TRPV4) channel. We previously found that TRPV4 physically and functionally interacts with AQP2 in cortical collecting ducts (CCD) cells, favoring cell volume regulation and cell migration. Because TRPV4 was implicated in ATP release in several tissues, we investigated the possibility that TRPV4/AQP2 interaction influences ATP release in CCD cells. Using two CCD cell lines expressing or not AQP2, we measured extracellular ATP (ATPe) under TRPV4 activation and intracellular Ca2+ under ATP addition. We found that AQP2 is critical for the release of ATP induced by TRPV4 activation. This ATP release occurs by an exocytic and a conductive route. ATPe, in turn, stimulates purinergic receptors leading to ATPe-induced ATP release by a Ca2+ -dependent mechanism. We propose that AQP2 by modulating Ca2+ and ATP differently could explain AQP2-increased cell migration.

Beyond Hot and Spicy: TRPV Channels and their Pharmacological Modulation.

Transient receptor potential vanilloid (TRPV) channels are part of the TRP channel superfamily and named after the first identified member TRPV1, that is sensitive to the vanillylamide capsaicin. Their overall structure is similar to the structure of voltage gated potassium channels (Kv) built up as homotetramers from subunits with six transmembrane helices (S1-S6). Six TRPV channel subtypes (TRPV1-6) are known, that can be subdivided into the thermoTRPV (TRPV1-4) and the Ca2+-selective TRPV channels (TRPV5, TRPV6). Contrary to Kv channels, TRPV channels are not primary voltage gated. All six channels have distinct properties and react to several endogenous ligands as well as different gating stimuli such as heat, pH, mechanical stress, or osmotic changes. Their physiological functions are highly diverse and subtype as well as tissue specific. In many tissues they serve as sensors for different pain stimuli (heat, pressure, pH) and contribute to the homeostasis of electrolytes, the maintenance of barrier functions and the development of macrophages. Due to their fundamental role in manifold physiological and pathophysiological processes, TRPV channels are promising targets for drug development. However, drugs targeting specific TRPV channels, that are suitable for drug therapy, are rare. Moreover, selective and potent compounds for further research at TRPV channels are often lacking. In this review different aspects of the structure, the different gating stimuli, the expression pattern, the physiological and pathophysiological roles as well as the modulating mechanisms of synthetic, natural and endogenous ligands are summarized.

Sweet scent lactones activate hot capsaicin receptor, TRPV1.

In this study, we investigated the activation of Transient receptor potential vanilloid subtype 1, TRPV1, by lactones, a representative flavor ingredient currently used for foods and beverages. As a result, we found that some lactones having C4 acyl chain length, γ-octalactone, δ-nonalactone and ß-methyl-γ-octalactone, γ-undecalactone with C7 acyl chain length and δ-undecalactone with C6 acyl chain length activated TRPV1. TRPV1 is known as a non-selective cation channels that respond to a wide range of physical and chemical stimuli such as high temperature, protons, capsaicin and so on. Furthermore, it has been also demonstrated that activation of TRPV1 induced energy expenditure enhancement and thermogenesis, suppressed accumulation of visceral fat in mice and prevented non-alcoholic fatty acid liver. Thus, lactones that function as TRPV1 agonists are thought to be important candidates for decreasing the risks of developing a metabolic syndrome.

TRPV1 activation inhibits phenotypic switching and oxidative stress in vascular smooth muscle cells by upregulating PPARα.

The proliferation and migration of vascular smooth muscle cells (VSMCs) is one of main reasons of vascular remodeling and is the pathogenesis of atherosclerosis and other vascular diseases. Transient receptor potential vanilloid 1 (TRPV1) is the specific receptor of capsaicin. TRPV1 has been previously reported to inhibit proliferation, migration and phenotypic switching, but the regulatory mechanisms and relevant signalling pathways are not clear. The aim of this study was to investigate the effects of capsaicin-activated TRPV1 on VSMC phenotypic switching. In this study, oxidized low density lipoprotein (ox-LDL) was used to induce the proliferation and migration of VSMCs. Our data showed that the VSMC proliferation induced by ox-LDL was dependent on the concentration of ox-LDL. Nevertheless, the data showed that capsaicin activated TRPV1 significantly decreased ox-LDL-induced superoxide anion generation. Phenotypic switching of VSMCs was inhibited by the activation of TRPV1. Furthermore, capsaicin decreased ox-LDL-induced superoxide anion generation by activating peroxisome proliferator activated receptor α (PPARα). TRPV1 inhibited VSMC phenotypic switching via upregulated expression of PPARα. It may be considered a useful target for the treatment of vascular remodeling.

The Pharmacological Case for Cannabigerol.

Medical cannabis and individual cannabinoids, such as Δ9-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD), are receiving growing attention in both the media and the scientific literature. The Cannabis plant, however, produces over 100 different cannabinoids, and cannabigerol (CBG) serves as the precursor molecule for the most abundant phytocannabinoids. CBG exhibits affinity and activity characteristics between Δ9-THC and CBD at the cannabinoid receptors but appears to be unique in its interactions with α-2 adrenoceptors and 5-hydroxytryptamine (5-HT1A). Studies indicate that CBG may have therapeutic potential in treating neurologic disorders (e.g., Huntington disease, Parkinson disease, and multiple sclerosis) and inflammatory bowel disease, as well as having antibacterial activity. There is growing interest in the commercial use of this unregulated phytocannabinoid. This review focuses on the unique pharmacology of CBG, our current knowledge of its possible therapeutic utility, and its potential toxicological hazards. SIGNIFICANCE STATEMENT: Cannabigerol is currently being marketed as a dietary supplement and, as with cannabidiol (CBD) before, many claims are being made about its benefits. Unlike CBD, however, little research has been performed on this unregulated molecule, and much of what is known warrants further investigation to identify potential areas of therapeutic uses and hazards.

Mechanosensitive TRPV4 is required for crystal-induced inflammation.

Crystal structures activate innate immune cells, especially macrophages and initiate inflammatory responses. We aimed to understand the role of the mechanosensitive TRPV4 channel in crystal-induced inflammation. Real-time RT-PCR, RNAscope in situ hybridisation, and Trpv4eGFP mice were used to examine TRPV4 expression and whole-cell patch-clamp recording and live-cell Ca2+ imaging were used to study TRPV4 function in mouse synovial macrophages and human peripheral blood mononuclear cells (PBMCs). Both genetic deletion and pharmacological inhibition approaches were used to investigate the role of TRPV4 in NLRP3 inflammasome activation induced by diverse crystals in vitro and in mouse models of crystal-induced pain and inflammation in vivo. TRPV4 was functionally expressed by synovial macrophages and human PBMCs and TRPV4 expression was upregulated by stimulation with monosodium urate (MSU) crystals and in human PBMCs from patients with acute gout flares. MSU crystal-induced gouty arthritis were significantly reduced by either genetic ablation or pharmacological inhibition of TRPV4 function. Mechanistically, TRPV4 mediated the activation of NLRP3 inflammasome by diverse crystalline materials but not non-crystalline NLRP3 inflammasome activators, driving the production of inflammatory cytokine interleukin-1ß which elicited TRPV4-dependent inflammatory responses in vivo. Moreover, chemical ablation of the TRPV1-expressing nociceptors significantly attenuated the MSU crystal-induced gouty arthritis. In conclusion, TRPV4 is a common mediator of inflammatory responses induced by diverse crystals through NLRP3 inflammasome activation in macrophages. TRPV4-expressing resident macrophages are critically involved in MSU crystal-induced gouty arthritis. A neuroimmune interaction between the TRPV1-expressing nociceptors and the TRPV4-expressing synovial macrophages contributes to the generation of acute gout flares.

Type I diabetes suppresses intracellular calcium ion increase normally evoked by heat stress in rat skeletal muscle.

Heat stress, via its effects on muscle intracellular Ca2+ concentrations ([Ca2+]i), has been invoked as a putative therapeutic countermeasure to type 1 diabetes-induced muscle atrophy. Using a circulation- and neurally intact in vivo muscle preparation, we tested the hypothesis that impaired muscle Ca2+ homeostasis in type 1 diabetic rats is due to attenuated heat stress tolerance mediated via transient receptor potential vanilloid 1 (TRPV1). Male Wistar rats were randomly assigned to one of the following four groups: 1) healthy control 30°C (CONT 30°C); 2) CONT 40°C; 3) diabetes 30°C (DIA 30°C); and 4) DIA 40°C. The temperature of 40°C was selected because it exceeds the TRPV1 activation threshold. Spinotrapezius muscles of Wistar rats were exteriorized in vivo and loaded with the fluorescent Ca2+ probe Fura-2 AM. [Ca2+]i was estimated over 20 min using fluorescence microscopy (340/380 nm ratio) in quiescent muscle held at the required temperature, using a calibrated heat source applied to the ventral muscle surface. Western blotting was performed to determine the protein expression levels of TRPV1 in spinotrapezius muscle. After 20 min of heat stress, the CONT 40°C condition induced a 12.3 ± 5% [Ca2+]i (P < 0.05) elevation that was markedly absent in the DIA 40°C or other conditions. Thus, no significant differences were found among DIA 40°C, DIA 30°C, and CONT 30°C. TRPV1 protein expression was decreased by 42.0 ± 9% in DIA compared with CONT (P < 0.05) and, unlike CONT, heat stress did not increase TRPV1 phosphorylation. In conclusion, diabetes suppresses TRPV1 protein expression and function and inhibits the elevated myocyte [Ca2+]i evoked normally by heat stress. These results suggest that capsaicin or other therapeutic strategies to increase Ca2+ accumulation via TRPV1 might be more effective than hyperthermic therapy for type 1 diabetic patients.

Activation of TRPV4 by mechanical, osmotic or pharmaceutical stimulation is anti-inflammatory blocking IL-1ß mediated articular cartilage matrix destruction.

OBJECTIVE: Cartilage health is maintained in response to a range of mechanical stimuli including compressive, shear and tensile strains and associated alterations in osmolality. The osmotic-sensitive ion channel Transient Receptor Potential Vanilloid 4 (TRPV4) is required for mechanotransduction. Mechanical stimuli inhibit interleukin-1ß (IL-1ß) mediated inflammatory signalling, however the mechanism is unclear. This study aims to clarify the role of TRPV4 in this response. DESIGN: TRPV4 activity was modulated glycogen synthase kinase (GSK205 antagonist or GSK1016790 A (GSK101) agonist) in articular chondrocytes and cartilage explants in the presence or absence of IL-1ß, mechanical (10% cyclic tensile strain (CTS), 0.33 Hz, 24hrs) or osmotic loading (200mOsm, 24hrs). Nitric oxide (NO), prostaglandin E2 (PGE2) and sulphated glycosaminoglycan (sGAG) release and cartilage biomechanics were analysed. Alterations in post-translational tubulin modifications and primary cilia length regulation were examined. RESULTS: In isolated chondrocytes, mechanical loading inhibited IL-1ß mediated NO and PGE2 release. This response was inhibited by GSK205. Similarly, osmotic loading was anti-inflammatory in cells and explants, this response was abrogated by TRPV4 inhibition. In explants, GSK101 inhibited IL-1ß mediated NO release and prevented cartilage degradation and loss of mechanical properties. Upon activation, TRPV4 cilia localisation was increased resulting in histone deacetylase 6 (HDAC6)-dependent modulation of soluble tubulin and altered cilia length regulation. CONCLUSION: Mechanical, osmotic or pharmaceutical activation of TRPV4 regulates HDAC6-dependent modulation of ciliary tubulin and is anti-inflammatory. This study reveals for the first time, the potential of TRPV4 manipulation as a novel therapeutic mechanism to supress pro-inflammatory signalling and cartilage degradation.

Transient receptor potential vanilloid four channels modulate inhibitory inputs through differential regulation of GABA and glycine receptors in rat retinal ganglion cells.

The transient receptor potential vanilloid 4 (TRPV4) channel is widely distributed in the retina. Activation of the TRPV4 channel enhances excitatory signaling from bipolar cells to retinal ganglion cells (RGCs), thereby increasing RGC firing rate and membrane excitability. In this study, we investigated the effect of TRPV4 channel activation on the miniature inhibitory postsynaptic current (mIPSC) in rat RGCs. Our results showed that perfusion with HC-067047, a TRPV4-channel antagonist, significantly reduced the amplitude of RGC mIPSCs. Extracellular application of the TRPV4 channel agonist GSK1016790A (GSK101) enhanced the frequency and amplitude of mIPSCs in ON- and OFF-type RGCs; pre-application of HC-067047 blocked the effect of GSK101 on mIPSCs. Furthermore, TRPV4 channels were able to enhance the frequency and amplitude of glycine receptor (GlyR)-mediated mIPSCs and inhibit the frequency of type A γ-aminobutyric acid receptor (GABAA R)-mediated mIPSCs. Upon intracellular administration or intravitreal injection of GSK101, TRPV4 channel activation reduced the release of presynaptic glycine and enhanced the function and expression of postsynaptic GlyRs; however, it inhibited presynaptic release of GABA, but did not affect postsynaptic GABAA Rs. Our study results provide insight regarding the effect of TRPV4 channel activation on RGCs and offer a potential interventional target for retinal diseases involving TRPV4 channels.