Activation Of Transient Receptor Potential Vanilloid Research Articles

TRPV4-induced inflammatory response is involved in neuronal death in pilocarpine model of temporal lobe epilepsy in mice

Activation of transient receptor potential vanilloid 4 (TRPV4) induces neuronal injury. TRPV4 activation enhances inflammatory response and promotes the proinflammatory cytokine release in various types of tissue and cells. Hyperneuroinflammation contributes to neuronal damage in epilepsy. Herein, we examined the contribution of neuroinflammation to TRPV4-induced neurotoxicity and its involvement in the inflammation and neuronal damage in pilocarpine model of temporal lobe epilepsy in mice. Icv. injection of TRPV4 agonist GSK1016790A (GSK1016790A-injected mice) increased ionized calcium binding adapter molecule-1 (Iba-1) and glial fibrillary acidic protein (GFAP) protein levels and Iba-1-positive (Iba-1+) and GFAP-positive (GFAP+) cells in hippocampi, which indicated TRPV4-induced microglial cell and astrocyte activation. The protein levels of nucleotide-binding oligomerization domain-like receptor pyrin domain containing 3 (NLRP3) inflammasome components NLRP3, apoptosis-related spotted protein (ASC) and cysteinyl aspartate-specific protease-1 (caspase-1) were increased in GSK1016790A-injected mice, which indicated NLRP3 inflammasome activation. GSK1016790A also increased proinflammatory cytokine IL-1β, TNF-α and IL-6 protein levels, which were blocked by caspase-1 inhibitor Ac-YVAD-cmk. GSK1016790A-induced neuronal death was attenuated by Ac-YVAD-cmk. Icv. injection of TRPV4-specific antagonist HC-067047 markedly increased the number of surviving cells 3 d post status epilepticus in pilocarpine model of temporal lobe epilepsy in mice (pilocarpine-induced status epilepticus, PISE). HC-067047 also markedly blocked the increase in Iba-1 and GFAP protein levels, as well as Iba-1+ and GFAP+ cells 3 d post-PISE. Finally, the increased protein levels of NLRP3, ASC and caspase-1 as well as IL-1β, TNF-α and IL-6 were markedly blocked by HC-067047. We conclude that TRPV4-induced neuronal death is mediated at least partially by enhancing the neuroinflammatory response, and this action is involved in neuronal injury following status epilepticus.

Expression and Activity of TRPA1 and TRPV1 in the Intervertebral Disc: Association with Inflammation and Matrix Remodeling.

Transient receptor potential (TRP) channels have emerged as potential sensors and transducers of inflammatory pain. The aims of this study were to investigate (1) the expression of TRP channels in intervertebral disc (IVD) cells in normal and inflammatory conditions and (2) the function of Transient receptor potential ankyrin 1 (TRPA1) and Transient receptor potential vanilloid 1 (TRPV1) in IVD inflammation and matrix homeostasis. RT-qPCR was used to analyze human fetal, healthy, and degenerated IVD tissues for the gene expression of TRPA1 and TRPV1. The primary IVD cell cultures were stimulated with either interleukin-1 beta (IL-1β) or tumor necrosis factor alpha (TNF-α) alone or in combination with TRPA1/V1 agonist allyl isothiocyanate (AITC, 3 and 10 µM), followed by analysis of calcium flux and the expression of inflammation mediators (RT-qPCR/ELISA) and matrix constituents (RT-qPCR). The matrix structure and composition in caudal motion segments from TRPA1 and TRPV1 wild-type (WT) and knock-out (KO) mice was visualized by FAST staining. Gene expression of other TRP channels (A1, C1, C3, C6, V1, V2, V4, V6, M2, M7, M8) was also tested in cytokine-treated cells. TRPA1 was expressed in fetal IVD cells, 20% of degenerated IVDs, but not in healthy mature IVDs. TRPA1 expression was not detectable in untreated cells and it increased upon cytokine treatment, while TRPV1 was expressed and concomitantly reduced. In inflamed IVD cells, 10 µM AITC activated calcium flux, induced gene expression of IL-8, and reduced disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5) and collagen 1A1, possibly via upregulated TRPA1. TRPA1 KO in mice was associated with signs of degeneration in the nucleus pulposus and the vertebral growth plate, whereas TRPV1 KO did not show profound changes. Cytokine treatment also affected the gene expression of TRPV2 (increase), TRPV4 (increase), and TRPC6 (decrease). TRPA1 might be expressed in developing IVD, downregulated during its maturation, and upregulated again in degenerative disc disease, participating in matrix homeostasis. However, follow-up studies with larger sample sizes are needed to fully elucidate the role of TRPA1 and other TRP channels in degenerative disc disease.

Functional studies of TRPV4 in Choroid Plexus Epithelial Cells

Cerebrospinal fluid (CSF) is produced by the choroid plexus epithelial (CPe) cells, and is moved throughout the brain ventricular system in coordinated waves that are generated by ependymal cilia lining the ventricles and subsequently absorbed by the subarachnoid granulations. CSF is moved in a pulsatile motion which is thought to be influenced by multiple factors like blood pressure and circadian rhythms. Hydrocephalus is a blanket disease description which covers clinical manifestations from CSF over‐production to decreased absorption and can occur at any age. Patients with pediatric hydrocephalus have enlarged ventricles, failure to thrive, sleepiness, sunsetting of the eyes, and vomiting. The current treatment paradigm for hydrocephalus is exclusively surgical and includes CSF diversion by shunt or endoscopic third ventriculostomy. These surgeries have known morbidity and mortality rates, and variable patient outcomes. In order to study pediatric hydrocephalus, the lab has two mouse lines which develop hydrocephalus postnatally. These models have been backcrossed to different genetic backgrounds in order to characterize disease severity, and to select the models with the most viability to undergo further studies. Disease severity is characterized by ventricular area as a function of total brain area in Nissl stained brain slices at specific points from the bregma. Additional characterization is being performed by utilizing intracranial pressure (ICP) monitoring using a small probe inserted into the brain parenchyma. The models that are the most stable and have disease severity similar to pediatric hydrocephalus will be used to test a class of drugs known as Transient Receptor Potential Vanilloid 4 (TRPV4) antagonists. TRPV4 is a widely expressed, widely translated protein which functions in many epithelial tissues as well as in the vasculature, and several neuronal cell types. TRPV4 can function as an osmosensor, mechanosensor, or thermosensor, and can be activated by a number of different metabolites. Activation of TRPV4 results in cation influx into the epithelial cells which can secondarily affect other transporters. TRPV4 antagonists have been shown to ameliorate hydrocephalus in the rat model of pediatric hydrocephalus, the WPK rat. To further understand the in vivo mechanism of TRPV4, a CPe line (Porcine Choroid Plexus – Riems) was used to characterize the effects of pressure (both chronic and acute), and shear force on the TRPV4‐mediated ion flux and conductance. TRPV4‐mediated ion flux is potentiated by acute pressure on the apical side of the CPe cells. In addition, both chronic and acute pressure and shear stress increase TRPV4‐mediated conductance. Successfully completing drug studies in the mouse models of hydrocephalus, as well as characterizing the mechanism of action of the TRPV4 antagonist drugs in vivo will provide a solid basis for further preclinical pharmacology studies.Support or Funding InformationGrants from the Hydrocephalus Association and the Department of Defense Office of the Congressionally Directed Medical Research Programs (CDMRP).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Transient Receptor Potential Vanilloid 4 channels participate in mouse ventricular action potentia

Transient Receptor Potential Vanilloid 4 (TRPV4) channels, belonging to the TRP channels superfamily, are Ca 2+ -permeable channels activated by hypo-osmotic stress. In the heart, TRPV4 channels are expressed at the protein level in mouse and rat ventricular myocytes and fibroblasts. They are involved in valve development during embryogenesis, in ischemia-reperfusion injuries and in reactive fibrosis. Whether TRPV4 channel can participate in ventricular action potential (AP) is still unknown. The aim of this study is to pharmacologically unmask the contribution of TRPV4 channels in left ventricular AP. Left Ventricular myocytes were isolated from TRPV4 + / + and TRPV4 − / − mice and AP were recorded using the patch-clamp technique. Pharmacological activator (GSK1016790A) and inhibitors (HC-067047 and GSK2193874) were perfused during the recordings to evaluate the effect on AP parameters. TRPV4 activator GSK1016790A induces a transient and dose dependent increase in AP duration at 90% of repolarization (APD90) in TRPV4 + / + but not in TRPV4 − / − myocytes (+ 19.4 ± 3.8%; n = 11 vs − 0.6 ± 1.8%; n = 7 at 500 nM respectively). The activator mediated-APD increase is abolished by TRPV4 channels inhibitors and CamKII inhibitor KN93 at 1 μM. Conversely, TRPV4 channel inhibitor (GSK2193874) at 100 nM significantly decreases APD90 in TRPV4 + / + but not in TRPV4 − / − myocytes (− 7.8 ± 2.6%; n = 11 vs 1.0 ± 1.3%; n = 7 respectively). Similarly, HC-067047 at 300 nM significantly decreases APD90 in TRPV4 + / + but not in TRPV4 − / − myocytes (− 10.9 ± 5.0%; n = 6 vs − 1.2 ± 2.4%; n = 4 respectively). At the concentrations mentioned above, activator and inhibitors have no effect on the other AP parameters. This study shows that TRPV4 channels exert a basal contribution in ventricular AP and could affect repolarization. It is thus a potential target for pharmacological approaches against ventricular arrhythmias.

Immunomodulatory effect of oleoylethanolamide in dendritic cells via TRPV1/AMPK activation.

Oleoylethanolamide (OEA) is an endogenous lipid mediator involved in the control of feeding, body weight, and energy metabolism. However, whether OEA modulates maturation of dendritic cells (DCs) has never been addressed. Hence, we evaluated the effect of OEA on DCs maturation in bone marrow-derived DCs (BMDCs) in four aspects: (a) Cell surface markers were determined using flow cytometric analysis; (b) cell mobile ability was testified with the transwell assay; (c) stimulation of T cells proliferation was performed in a coculture system; and (d) cytokine production was measured using polymerase chain reaction (PCR). The result showed that, in mature BMDCs induced by lipopolysaccharides (LPS), the OEA treatment decreased expressions of cell surface markers, reduced cell migration, diminished the proliferation of cocultured T cells, and regulated cytokine production of BMDCs, indicating the modulatory effect of OEA on DCs maturation. Furthermore, to explore the underlying mechanism of the immunomodulatory effect of OEA, we used antagonists of transient receptor potential vanilloid-1 (TRPV1) and AMP-activated protein kinase (AMPK), determined the protein expressions of TRPV1/AMPK and Toll-like receptor 4 (TLR4)/nuclear factor-kappa B (NF-κB) using western blot, and measured the intracellular calcium concentration using calcium imaging. The result illustrated that OEA downregulated TLR4/NF-κB, the classical pathway leading to DCs maturation induced by LPS, through the activation of TRPV1 and AMPK. Collectively, the present study suggests that OEA suppresses DCs maturation through the activation of TRPV1/AMPK. These findings increase our understanding of this endogenous lipid OEA.

Molecular and functional insights into Transient Receptor Potential Vanilloid 1 (TRPV1) in bull spermatozoa

In view of the limited information available on functional significance of TRPV1 in regulating sperm functions, present study was undertaken on bull spermatozoa. Sixty four ejaculates were collected from four Hariana bulls and were used for molecular and functional characterisation of TRPV1. Immunoblotting using TRPV1 specific antibody revealed the presence of a single band of 104 kDa corresponding to TRPV1 in Hariana bull spermatozoa. Indirect immuno fluorescence revealed positive immune-reactivity to TRPV1 at acrosomal, pre-acrosomal, post acrosomal and flagellar regions of spermatozoa. Based on the results of pilot study dose-response analysis, doses of anandamide (AEA; 0.3 μM) and capsazepine (Cp; 10 μM) were selected for further studies. Three groups of semen samples (control 100 μL diluted semen having 1 × 106 spermatozoa; anandamide (3 μL AEA+97 μL of diluted semen containing 1 × 106 spermatozoa and Cp (1 μL Cp+99 μL of diluted semen containing 1 × 106 spermatozoa) were used to study the functional involvement of TRPV1 in bull spermatozoa. Blocking of TRPV1 with Cp resulted in significant (P < 0.05) reduction in progressive sperm motility (PSM) as compared to control. With activation of TRPV1 using AEA also, PSM was significantly (P < 0.05) decreased till 1h and thereafter the PSM was sustained to the level as observed in control. However, both during blocking and activation of TRPV1, per cent spermatozoa showing hyperactive motility were significantly (P < 0.05) increased (20–30%) compared to the control. Treatment with both Cp and AEA revealed significant (P < 0.05) increase in B-pattern of spermatozoa in chlortetracycline hydrochloride (CTC) staining indicating induction of capacitation. Inhibition of soluble adenyl cyclase (sAC) with 99 nM KH7and protein kinase A (PKA) with 3 μM P9115 significantly (P < 0.05) decreased PSM both in the presence of Cp and AEA. Blocking as well as activation of TRPV1 showed significant (P < 0.05) reduction in sperm livability, intact membrane, intact acrosome, high mitochondrial transmembrane potential; hence indicating the involvement of TRPV1 in regulation of sperm functions in bulls. From the study-it was concluded that TRPV1 channels are found in bull spermatozoa and mediate number of sperm functions like motility, hypermotility, capacitation and acrosome reaction. Further studies are required to find out the possible relationship between TRPV1 channels and other channels in regulating spermatozoa function and possible mechanisms associated with TRPV1 activation as well as its role in sperm function regulation.

Phosphatidylinositol Inhibits TRPV1 via its Vanilloid Binding Site

Regulation of the heat- and capsaicin-activated Transient Receptor Potential Vanilloid 1 (TRPV1) channels by phosphoinositides is complex and controversial. In excised inside-out patches phosphatidylinositol 4,5-bisphosphate (PIP2) and its precursor phosphatidylinositol 4-phosphate (PIP) reproducibly potentiate TRPV1 activity. In a recent cryoEM structure a phosphoinositide was detected in the vanilloid binding site in the absence of agonists. Phosphoinositides were proposed to negatively regulate TRPV1 as competitive inhibitors of vanilloids, but the resolution of the structure was sufficient to determine the exact nature of the lipid. Here we used computational modeling, site directed mutagenesis and patch clamp electrophysiology to identify the phosphoinositide in the vanilloid binding site. We find that PIP2 diffuses away from the vanilloid binding site in Molecular Dynamics simulations, but phosphatidylinositol, the precursor of PIP and PIP2, remains there. We also find that mutating two residues predicted to interact with phosphatidylinositol, but not with capsaicin shifts the dose-response of capsaicin to the left. We also show that in excised inside-out patch clamp experiments phosphatidylinositol inhibited TRPV1 activity induced by PIP2 at low capsaicin concentrations. We conclude that phosphatidylinositol binds to the Vanilloid binding site of TRPV1 in resting conditions, and this may serve to set the basal sensitivity of the channel to vanilloids as well as heat and low pH.

TRPV1 channel contributes to remifentanil-induced postoperative hyperalgesia via regulation of NMDA receptor trafficking in dorsal root ganglion.

BackgroundRemifentanil is widely used in general anesthesia due to its reliability and rapid onset. However, remifentanil-induced postoperative hyperalgesia might be a challenge nowadays. Accumulating evidence suggests that the transient receptor potential vanilloid 1 (TRPV1) was involved in the development of neuropathic pain and hyperalgesia. However, the contribution of TRPV1 in modulating remifentanil-induced postoperative hyperalgesia is still unknown. The aim of this study is the contribution of TRPV1 to the surface expression of N-methyl-d-aspartate (NMDA) receptors in remifentanil-induced postoperative hyperalgesia.MethodsThe hot plate test and the Von Frey test were performed to evaluate thermal and mechanical hyperalgesia. Capsazepine (CPZ) was administrated intrathecally to confirm our results. TRPV1, NMDA receptors, CaMKII (calcium/calmodulin-dependent kinase II), and protein kinase C (PKC) in the dorsal root ganglion (DRG) were detected by Western blotting. Immunofluorescence assay was applied to analyze the distribution of TRPV1 and the relationship between TRPV1 and NMDA receptor subunit 1 (NR1).ResultsRemifentanil-induced both thermal and mechanical postoperative hyperalgesia. Here, we found the membrane trafficking of NR1, possibly due to the activation of TRPV1 in DRG neurons after remifentanil infusion. Furthermore, intrathecal injection of CPZ was able to relieve remifentanil-induced postoperative hyperalgesia according to a behavioral test and CPZ confirmed that TRPV1 is involved in NR1 trafficking. In addition, CaMKII/PKC but not protein kinase A (PKA) contributed to remifentanil-induced postoperative hyperalgesia.ConclusionOur study demonstrates that TRPV1 receptors are involved in remifentanil-induced postoperative hyperalgesia. TRPV1 contributes to the persistence of remifentanil-induced postoperative hyperalgesia through the trafficking of NMDA receptors via the activation of CaMKII-PKC signaling pathways in DRG neurons.

TRPV4-mediated calcium signaling in mesenchymal stem cells regulates aligned collagen matrix formation and vinculin tension

Microarchitectural cues drive aligned fibrillar collagen deposition in vivo and in biomaterial scaffolds, but the cell-signaling events that underlie this process are not well understood. Utilizing a multicellular patterning model system that allows for observation of intracellular signaling events during collagen matrix assembly, we investigated the role of calcium (Ca2+) signaling in human mesenchymal stem cells (MSCs) during this process. We observed spontaneous Ca2+ oscillations in MSCs during fibrillar collagen assembly, and hypothesized that the transient receptor potential vanilloid 4 (TRPV4) ion channel, a mechanosensitive Ca2+-permeable channel, may regulate this signaling. Inhibition of TRPV4 nearly abolished Ca2+ signaling at initial stages of collagen matrix assembly, while at later times had reduced but significant effects. Importantly, blocking TRPV4 activity dramatically reduced aligned collagen fibril assembly; conversely, activating TRPV4 accelerated aligned collagen formation. TRPV4-dependent Ca2+ oscillations were found to be independent of pattern shape or subpattern cell location, suggesting this signaling mechanism is necessary for aligned collagen formation but not sufficient in the absence of physical (microarchitectural) cues that force multicellular alignment. As cell-generated mechanical forces are known to be critical to the matrix assembly process, we examined the role of TRPV4-mediated Ca2+ signaling in force generated across the load-bearing focal adhesion protein vinculin within MSCs using an FRET-based tension sensor. Inhibiting TRPV4 decreased tensile force across vinculin, whereas TRPV4 activation caused a dynamic unloading and reloading of vinculin. Together, these findings suggest TRPV4 activity regulates forces at cell-matrix adhesions and is critical to aligned collagen matrix assembly by MSCs.

Physiological and pathophysiological roles of TRPV4 channel in gastrointestinal tract

Transient receptor potential vanilloid 4 (TRPV4) is a non-selective cation channel that responds to mechanical, thermal, and chemical stimuli in addition to various endogenous ligands, such as arachidonic acid metabolites. The present study aimed to elucidate the expression of TRPV4 in the gastrointestinal tract and the pathogenic roles of TRPV4 in dextran sulphate sodium (DSS)-induced colitis. TRPV4-immunoreactivity was detected in epithelial-like cells of the mouse tongue, esophagus, stomach, ileum, and colon; TRPV4 expression in the tongue was higher than other gastrointestinal tracts. TRPV4 colocalized with a type IV cell marker sonic hedgehog in circumvallate papillae. These findings suggest that TRPV4 contributes to sour taste sensing by regulating type III taste cell differentiation in mice. DSS-induced colitis was significantly attenuated in TRPV4-knockout (TRPV4KO) mice when compared to wild-type mice. DSS treatment upregulated TRPV4 expression in vascular endothelia of colonic mucosa and submucosa. DSS treatment increased vascular permeability, which was abolished in TRPV4KO mice. The activation of TRPV4 decreased VE-cadherin expression in mouse aortic endothelial cells exposed to TNF-α. These findings indicate that the upregulation of TRPV4 in vascular endothelial cells contributes to the progression of colonic inflammation via the activation of vascular permeability. Thus, TRPV4 is an attractive target for the treatment of inflammatory bowel diseases.

Differences in the effects of TRPV1 antagonists on energy metabolism in mice.

Transient receptor potential vanilloid 1 (TRPV1) is a nociceptive cation channel that is activated by heat, protons and chemical ligands such as capsaicin. We investigated the roles of the capsaicin receptor, TRPV1, in controlling the energy metabolism of the whole body. It has been reported that the activation of TRPV1 by its agonists enhances energy metabolism. In this study, we used a respiratory gas analysis system to examine whether the inhibition of TRPV1 changes energy metabolism in mice. In addition, we examined the contributions of different modes of TRPV1 activation (heat, protons and capsaicin) to determine the influence of 3 different TRPV1 antagonists on energy metabolism. Here, we showed that intragastric administration of AMG517, a nonselective antagonist of TRPV1 (for heat, protons and capsaicin), enhanced energy metabolism as much as did intraperitoneal administration. On the other hand, intraperitoneal administration of AMG9810, a nonselective antagonist like AMG517, enhanced energy expenditure more than intragastric administration. However, the administration of JYL1421, a TRPV1 antagonist that very strongly inhibits TRPV1 activated by capsaicin, did not change energy metabolism. Taken together, these results suggest that the type of TRPV1 antagonists and the routes of its administration have different effects on energy metabolism in a normal body. Surprisingly, co-administration of JYL1421 and capsaicin significantly enhanced the energy metabolism more than administration of capsaicin alone. These results support the possibility that an unconventional mechanism is responsible for the increase in energy metabolism that occurs via TRPV1 inhibition.

Sirtuin 1 inhibits TNF-α-mediated osteoclastogenesis of bone marrow-derived macrophages through both ROS generation and TRPV1 activation.

Sirtuin 1 (SIRT1), also known as NAD-dependent deacetylase, has been reported to increase in vivo osteoclast-mediated bone resorption. However, its effects on osteoclastogenesis or bone loss in vitro have not been widely examined. Therefore, the effects and underlying mechanism of SIRT1 on osteoclast differentiation in mice in vitro were studied. During RANKL-induced osteoclastogenesis in differentiated bone marrow-derived macrophages (BMMs), SIRT1 downregulation was observed. The use of resveratrol (SIRT1 activator) and SIRT1 overexpression was found to inhibit osteoclastogenesis, which was confirmed by TRAP staining and activity loss, reduced expression of osteoclast markers and related genes, and a decrease in the number of multinuclear cells. In contrast, treatment with EX-527 (SIRT1 inhibitor) as well as SIRT1 silencing promoted osteoclastogenesis. Furthermore, the tumor necrosis factor (TNF)-α level was reduced by resveratrol treatment and SIRT1 overexpression but increased following EX-527 incubation and SIRT1 depletion. TNF-α silencing blocked the osteoclastogenesis of BMMs promoted by SIRT1 depletion. Moreover, transient receptor potential vanilloid 1 (TRPV1) channel activation and reactive oxygen species (ROS) production, which are associated with osteoclastogenesis, were impaired by TNF-α silencing. These data demonstrate that SIRT1 directly inhibits osteoclastogenesis by inhibiting ROS generation and TRPV1 channel activation under mediation of TNF-α.

TRPV4 Inhibition Improved Myelination and Reduced Glia Reactivity and Inflammation in a Cuprizone-Induced Mouse Model of Demyelination.

The inhibition of demyelination and the promotion of remyelination are both considerable challenges in the therapeutic process for many central nervous system (CNS) diseases. Increasing evidence has demonstrated that neuroglial activation and neuroinflammation are responsible for myelin sheath damage during demyelinating disorders. It has been revealed that the nonselective cation channel transient receptor potential vanilloid 4 (TRPV4) profoundly affects a variety of physiological processes, including inflammation. However, its roles and mechanisms in demyelination have remained unclear. Here, for the first time, we found that there was a significant increase in TRPV4 in the corpus callosum in a demyelinated mouse model induced by cuprizone (CPZ). RN-1734, a TRPV4-antagonist, clearly alleviated demyelination and inhibited glial activation and the production of tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β) without altering the number of olig2-positive cells. In vitro, RN-1734 treatment clearly inhibited the influx of calcium and decreased the levels of IL-1β and TNF-α in lipopolysaccharide (LPS)-activated microglial cells by suppressing NF-κB P65 phosphorylation. Apoptosis of oligodendrocyte induced by LPS-activated microglia was also alleviated by RN-1734. The results suggest that activation of TRPV4 in microglia is involved in oligodendrocyte apoptosis through the activation of the NF-κB signaling pathway, thus revealing a new mechanism of CNS demyelination.

Taurine Inhibits TRPV-Dependent Activity to Overcome Oxidative Stress in Caenorhabditis elegans.

Taurine has important physiological roles as well as a wide range of pharmacological effects. Studies have suggested that taurine ameliorates diabetes, hypertension, oxidative stress, and inflammatory diseases. However, its mechanisms of action are still unclear. It has been reported that N-acyl taurine activates transient receptor potential vanilloid-1 (TRPV1), which is related to the pathogenesis of many inflammatory diseases. In this study, we hypothesized that taurine has a regulatory effect on TRPV1 activation via N-acyl taurine. To evaluate this hypothesis, we assessed the calcium influx activated by a TRPV1 agonist in human keratinocyte (HaCaT) cells and paraquat-induced oxidative stress in Caenorhabditis elegans. Our results indicate that taurine inhibits TRPV-dependent activity to overcome oxidative stress in cultured cell lines and in C. elegans.

Involvement of VNUT-exocytosis in transient receptor potential vanilloid 4-dependent ATP release from gastrointestinal epithelium.

Adenosine triphosphate (ATP) modulates mechanosensitive vagal afferent nerves in the gastrointestinal tract. ATP is stored in secretory vesicles via the ATP transporter VNUT. Recently, the bisphosphate clodronate was reported to inhibit VNUT and was suggested to be a safe potent therapeutic option for chronic pain. Transient receptor potential vanilloid 4 (TRPV4) is activated by mechanical stimuli and some epoxyeicosatrienoic acids and becomes sensitized under inflammatory conditions. We have previously reported that TRPV4 and VNUT are expressed in mouse esophageal keratinocytes and that TRPV4 activation induces ATP release in gastric epithelial cells. Here we show the expression of TRPV4 and VNUT in normal human gastrointestinal cell derived cell lines (GES-1 and CCD 841) and in tissues from normal and VNUT-KO mice. TRPV4 agonists (GSK101 or 8,9-EET) induced an increase in cytosolic Ca2+ and/or current responses in mouse primary colonic epithelial cells and CCD 841 cells, but not in cells isolated from TRPV4-KO mice. TRPV4 agonists (GSK101 or 5.6-EET) also induced ATP release in GES-1 and CCD 841 cells, which could be blocked by the VNUT inhibitor, clodronate. Thus, VNUT inhibition with clodronate could represent a novel therapeutic option for visceral pain.

A1935 Modulation of TRPV4 by apigenin ameliorates renal fibrosis in DOCA salt hypertensive rats

Objectives: Hypertension contributes to chronic renal damage. Apigenin, an effective anti-hypertensive flavone, acts as antagonist of transient receptor potential vanilloid 4 (TRPV4). Here, we aimed to investigate whether apigenin ameliorates hypertension-induced renal fibrosis and its underlying mechanism. Methods: Male Sprague-Dawley rats, 2-month-old TRPV4 knockout mice (TRPV4−/−) and their wild-type littermates were used to established deoxycorticosterone acetate (DOCA)–salt-induced hypertension model and received 0.2% apigenin diet for 4 weeks. The tail-cuff blood pressure (BP) was measured. Renal fibrosis was assessed by Histological staining and protein levels of fibrotic markers TGF-β1, p-Smad2/3, CTGF, COL1a1, and COL3a1 detected by western blot. Expression of TRPV4 was analyzed in kidney and renal mesangial cell. Cytosolic free Ca2+ was measured by fluorospectrometers. Results: Apigenin signifcantly attenuated hypertension-induced renal fibrosis, which was accompanied by reduced expression of TGF-β1/Smad2/3 signaling proteins. The inhibitory effect of apigenin on renal fibrosis is independent of blood pressure reduction. Immunochemistry, cell-attached patch clamp and fluorescent Ca2+ imaging results indicated that TRPV4 was expressed and activated by apigenin in both the kidney and renal cells. Importantly, knockout of TRPV4 in mice abolished the beneficial effects of apigenin that were observed in the DOCA–salt hypertensive rats. Additionally, apigenin directly inhibited activation of the TGF-β1/Smad2/3 signaling pathway in different renal tissues through activation of TRPV4 regardless of the type of pro-fibrotic stimulus. Conclusion: Dietary apigenin ameliorates hypertension-induced renal fibrosis through directly inhibiting the TGF-β1/Smad2/3 pathway in a TRPV4-dependent manner.

Carboxamido steroids inhibit the opening properties of transient receptor potential ion channels by lipid raft modulation

Transient Receptor Potential (TRP) cation channels, like the TRP Vanilloid 1 (TRPV1) and TRP Ankyrin 1 (TRPA1), are expressed on primary sensory neurons. These thermosensor channels play a role in pain processing. We have provided evidence previously that lipid raft disruption influenced the TRP channel activation, and a carboxamido-steroid compound (C1) inhibited TRPV1 activation. Therefore, our aim was to investigate whether this compound exerts its effect through lipid raft disruption and the steroid backbone (C3) or whether altered position of the carboxamido group (C2) influences the inhibitory action by measuring Ca2+ transients on isolated neurons and calcium-uptake on receptor-expressing CHO cells. Membrane cholesterol content was measured by filipin staining and membrane polarization by fluorescence spectroscopy. Both the percentage of responsive cells and the magnitude of the intracellular Ca2+ enhancement evoked by the TRPV1 agonist capsaicin were significantly inhibited after C1 and C2 incubation, but not after C3 administration. C1 was able to reduce other TRP channel activation as well. The compounds induced cholesterol depletion in CHO cells, but only C1 induced changes in membrane polarization. The inhibitory action of the compounds on TRP channel activation develops by lipid raft disruption, and the presence and the position of the carboxamido group is essential.

TRPV1 mediates capsaicin-stimulated metabolic activity but not cell death or inhibition of interleukin-1β release in human THP-1 monocytes

Human monocytes and dendritic cells express transient receptor potential vanilloid 1 (TRPV1) which may play a role in mediating the inflammatory, immune and cancer surveillance responses of these cells. The aim of the present study was to investigate TRPV1 expression and function in THP-1 monocytic cells. RT-PCR and Western blot were used to detect TRPV1. The metabolic activity and viability of THP-1 cells following exposure to vanilloids was assessed using resorufin production from rezazurin. Cytokine release was measured using ELISA. TRPV1 was expressed in cultured THP-1 monocytic cells and naïve monocytes. Lower concentrations (<250 μM) of capsaicin, but not other putative TRPV1 agonists, were shown to stimulate cell metabolic activity, whereas at concentrations >250 μM, all agonists decreased metabolic activity. Capsaicin-stimulated THP-1 metabolic activity was blocked by the TRPV1 antagonist, 5-iodo-resiniferatoxin (5′-IRTX), whereas the decline in resorufin production by THP-1 cells at higher capsaicin concentrations (due to cell death), was not affected by 5′-IRTX. Finally, capsaicin (≤125 μM) significantly increased lipopolysaccharide-stimulated IL-6 and TNF-α release from THP-1 cells, whereas phytohaemagglutinin-stimulated IL-1β, TNF-α, MCP-1 and IL-6 release were concentration-dependently inhibited by capsaicin. Modulation of IL-1β release was not TRPV1 mediated. Overall, these results show that functional TRPV1 channels are present in naïve monocytes and THP-1 cells, and when activated, increase cell metabolic activity. In addition, capsaicin modifies cytokine release from THP-1 cells and induces cell death, most likely by a mechanism that is independent of TRPV1 activation.

Retinal Detachment-Induced Müller Glial Cell Swelling Activates TRPV4 Ion Channels and Triggers Photoreceptor Death at Body Temperature.

Using region-specific injection of hyaluronic acid, we developed a mouse model of acute retinal detachment (RD) to investigate molecular mechanisms of photoreceptor cell death triggered by RD. We focused on the transient receptor potential vanilloid 4 (TRPV4) ion channel, which functions as a thermosensor, osmosensor, and/or mechanosensor. After RD, the number of apoptotic photoreceptors was reduced by ∼50% in TRPV4KO mice relative to wild-type mice, indicating the possible involvement of TRPV4 activation in RD-induced photoreceptor cell death. Furthermore, TRPV4 expressed in Müller glial cells can be activated by mechanical stimuli caused by RD-induced swelling of these cells, resulting in release of the cytokine MCP-1, which is reported as a mediator of Müller glia-derived strong mediator for RD-induced photoreceptor death. We also found that the TRPV4 activation by the Müller glial swelling was potentiated by body temperature. Together, our results suggest that RD adversely impacts photoreceptor viability via TRPV4-dependent cytokine release from Müller glial cells and that TRPV4 is part of a novel molecular pathway that could exacerbate the effects of hypoxia on photoreceptor survival after RD.SIGNIFICANCE STATEMENT Identification of the mechanisms of photoreceptor death in retinal detachment is required for establishment of therapeutic targets for preventing loss of visual acuity. In this study, we found that TRPV4 expressed in Müller glial cells can be activated by mechanical stimuli caused by RD-induced swelling of these cells, resulting in release of the cytokine MCP-1, which is reported as a mediator of Müller glia-derived strong mediator for RD-induced photoreceptor death. We also found that the TRPV4 activation by the Müller glial swelling was potentiated by body temperature. Hence, TRPV4 inhibition could suppress cell death in RD pathological conditions and suggests that TRPV4 in Müller glial cells might be a novel therapeutic target for preventing photoreceptor cell death after RD.

Vagal TRPV1 activation exacerbates thermal hyperpnea and increases susceptibility to experimental febrile seizures in immature rats

Thermal hyperpnea, a pattern of breathing during hyperthermia that is characterized by an increase in tidal volume as well as breathing frequency, is known to lead to respiratory alkalosis. Thermal hyperpnea-induced respiratory alkalosis is linked to febrile seizures (FS). The heat-sensitive transient receptor potential vanilloid-1 (TRPV1) receptors are localized in, and implicated in the heat sensitivity of peripheral and central structures involved in the respiratory response to hyperthermia. We, therefore, hypothesize that TRPV1 activation increases susceptibility to experimental FS (EFS) in immature rats due to an exacerbated thermal hyperpnea. We found that peripheral, but not central TRPV1 activation had pro-convulsant effects. These pro-convulsant effects were associated with an increased rate of expired CO2 due to an exaggerated ventilatory response to hyperthermia. The TRPV1 antagonist, AMG-9810, and TRPV1 deletion abolished the pro-convulsant effects, while exposure to 5% CO2, bilateral vagotomy and DREADD (designer receptor exclusively activated by designer drugs)-mediated inhibition of TRPV1-containing cells in the vagal nodose ganglia significantly attenuated these effects. These findings suggest that vagal TRPV1-driven thermal hyperpnea likely increases susceptibility to FS in immature rodents. This identifies a novel peripheral anatomical and molecular target that should be considered when developing therapeutics for FS.