Transient Receptor Potential Vanilloid Subfamily Research Articles

Do TRPV1 antagonists increase the risk for skin tumourigenesis? A collaborative in vitro and in vivo assessment.

A recent hypothesis suggesting that the pharmacological target TRPV1 (transient receptor potential vanilloid subfamily, member 1) may function as a tumour suppressor, which potentially impacts the development of TRPV1 antagonist therapeutics for a range of conditions. However, little is known about the long-term physiologic effects of TRPV1 blockade in the skin. In vitro and in vivo studies suggested that the potent TRPV1 competitive antagonist AMG-9810 promoted proliferation in N/TERT1 cells (telomerase-immortalised primary human keratinocytes 1) and tumour development in mouse skin that was mediated through EGFR/Akt/mTOR signalling. We attempted to reproduce the reported in vitro and in vivo findings to further explore this hypothesis to understand the underlying mechanism and the risk associated with TRPV1 antagonism in the skin. In vitro proliferation studies using multiple methods and topical application with AMG-9810 and structurally similar TRPV1 antagonists such as SB-705498 and PAC-14028 were performed. Although we confirmed expression of TRPV1 in primary human epidermal keratinocytes (HEKn) and spontaneously immortalised human keratinocytes (HaCaT), we were unable to demonstrate cell proliferation in either cell type or any clear evidence of increased expression of proteins in the EGFR/Akt/mTOR signalling pathway with these molecules. We were also unable to demonstrate skin tumour promotion or underlying molecular mechanisms involved in the EGFR/Akt/mTOR signalling pathway in a single-dose and two-stage carcinogenesis mouse study treated with TRPV1 antagonists. In conclusion, our data suggest that inhibiting the pharmacological function of TRPV1 in skin by specific antagonists has not been considered to be indicative of skin tumour development.

Measurement of Basal and Forskolin-stimulated Lipolysis in Inguinal Adipose Fat Pads.

Lipolysis is a process by which the lipid stored as triglycerides in adipose tissues are hydrolyzed into glycerol and fatty acids. This article describes the method for the measurement of basal and forskolin (FSK)-stimulated lipolysis in the inguinal fat pads isolated from wild type mice fed either normal chow diet (NCD), high fat diet (HFD) or a high fat diet containing 0.01% of capsaicin (CAP; transient receptor potential vanilloid subfamily 1 (TRPV1) agonist) for 32 weeks. The method described here for performing ex vivo lipolysis is adopted from Schweiger et al.1 We present a detailed protocol for measuring glycerol levels by UV-Visible (UV/VIS) spectrophotometry. The method described here can be used to successfully isolate inguinal fat pads for lipolysis measurements to obtain consistent results. The protocol described for inguinal fat pads can readily be extended to measure lipolysis in other tissues.

Direct Anandamide Activation of TRPV1 Produces Divergent Calcium and Current Responses

In the brainstem nucleus of the solitary tract (NTS), primary vagal afferent neurons express the transient receptor potential vanilloid subfamily member 1 (TRPV1) at their central terminals where it contributes to quantal forms of glutamate release. The endogenous membrane lipid anandamide (AEA) is a putative TRPV1 agonist in the brain, yet the extent to which AEA activation of TRPV1 has a neurophysiological consequence is not well established. We investigated the ability of AEA to activate TRPV1 in vagal afferent neurons in comparison to capsaicin (CAP). Using ratiometric calcium imaging and whole-cell patch clamp recordings we confirmed that AEA excitatory activity requires TRPV1, binds competitively at the CAP binding site, and has low relative affinity. While AEA-induced increases in peak cytosolic calcium were similar to CAP, AEA-induced membrane currents were significantly smaller. Removal of bath calcium increased the AEA current with no change in peak CAP currents revealing a calcium sensitive difference in specific ligand activation of TRPV1. Both CAP- and AEA-activated TRPV1 currents maintained identical reversal potentials, arguing against a major difference in ion selectivity to resolve the AEA differences in signaling. In contrast with CAP, AEA did not alter spontaneous glutamate release at NTS synapses. We conclude: (1) AEA activation of TRPV1 is markedly different from CAP and produces different magnitudes of calcium influx from whole-cell current; and (2) exogenous AEA does not alter spontaneous glutamate release onto NTS neurons. As such, AEA may convey modulatory changes to calcium-dependent processes, but does not directly facilitate glutamate release.

Inhibition of TNF reduces mechanical orofacial hyperalgesia induced by Complete Freund's Adjuvant by a TRPV1-dependent mechanism in mice.

Inflammation in the orofacial region results in pain and is associated with many pathological states, including migraine, neuralgias and temporomandibular disorder. Although extensively studied, the mechanisms responsible for these conditions are not known and effective treatments are lacking. We reported earlier that the proinflammatory cytokine tumor necrosis factor (TNF) plays an important role in regulation of trigeminal ganglion (TG) neuron function in vitro. In the present study we investigated the role of TNF in mechanical hypersensitivity in mice. We employed the Complete Freund's Adjuvant (CFA)-induced model of orofacial pain and evaluated the effect of blocking of soluble TNF activity by peripheral administration of the novel dominant negative TNF biologic, XPro1595. We show that CFA administration into the lower lip causes hyperalgesia and an increase in both expression of transient receptor potential vanilloid subfamily member 1 (TRPV1) mRNA and in the average intensity of TRPV1 protein immunoreactivity in TG neurons. We also show that intraperitoneal administration of XPro1595 prevents both CFA-induced mechanical hypersensitivity and, as shown in immunohistochemical staining - upregulation of TRPV1 protein expression in TG neurons. We conclude that one of the possible regulatory mechanisms of TNF in pain involves upregulation of the nociceptor TRPV1, and that peripheral treatment with a selective anti-soluble TNF biologic can prevent hyperalgesia caused by inflammation in the orofacial region. Therefore, these new findings suggest that XPro1595 may serve as a novel treatment for orofacial pain disorders.

Exploration of the Esophageal Mucosal Barrier in Non-Erosive Reflux Disease.

In the absence of visible mucosal damage, it is hypothesized that the esophageal mucosal barrier is functionally impaired in patients with non-erosive reflux disease (NERD). The aim of the present study was to perform an exploratory analysis of the mucosal barrier in NERD compared to erosive esophagitis (EE) and controls. A second aim was to explore TRPV1 gene transcription in relation to the mucosal barrier function and heartburn symptoms. In this prospective study, 10 NERD patients, 11 patients with active erosive esophagitis and 10 healthy volunteers were included. Biopsies from non-eroded mucosa were obtained for (1) ex vivo analyses (Ussing chamber) of transepithelial electrical resistance (TEER) and permeability (2) gene transcription of tight-junction proteins and transient receptor potential vanilloid subfamily member 1 (TRPV1). No differences in TEER or permeability were found between NERD and healthy volunteers, whereas TEER was lower in patients with erosive esophagitis. TRPV1 gene transcription was not significantly different between EE, NERD and controls. Conclusions: esophageal mucosal barrier function and TRPV1 transcription is not significantly altered in NERD patients. Future research is needed to explore other potential mechanisms that may account for the high symptom burden in these patients.

Transient receptor potential vanilloid 5 (TRPV5), a highly Ca2+ -selective TRP channel in the rat brain: relevance to neuroendocrine regulation.

Recent studies suggest an important role for transient receptor potential vanilloid (TRPV) ion channels in neural and neuroendocrine regulation. The TRPV subfamily consists of six members: TRPV1-6. While the neuroanatomical and functional correlates of TRPV1-4 have been studied extensively, relevant information about TRPV5 and TRPV6, which are highly selective for Ca2+ , is limited. We detected TRPV5 mRNA expression in the olfactory bulb, cortex, hypothalamus, hippocampus, midbrain, brainstem and cerebellum of the rat. TRPV5-immunoreactive neurones were conspicuously seen in the hypothalamic paraventricular (PVN), supraoptic (SON), accessory neurosecretory (ANS), supraoptic nucleus, retrochiasmatic part (SOR), arcuate (ARC) and medial tuberal nuclei, hippocampus, midbrain, brainstem and cerebellum. Glial cells also showed TRPV5-immunoreactivity. To test the neuroendocrine relevance of TRPV5, we focused on vasopressin, oxytocin and cocaine- and amphetamine-regulated transcript (CART) as representative candidate markers with which TRPV5 may co-exist. In the hypothalamic neurones, co-expression of TRPV5 was observed with vasopressin (PVN: 50.73±3.82%; SON: 75.91±2.34%; ANS: 49.12±4.28%; SOR: 100%) and oxytocin (PVN: 6.88±1.21; SON: 63.34±5.69%; ANS: 20.4±4.14; SOR: 86.5±1.74%). While ARC neurones express oestrogen receptors, 17β-oestradiol regulates TRPV5, as well as CART neurones and astrocytes, in the ARC. Furthermore, ARC CART neurones are known to project to the preoptic area, and innervate and regulate GnRH neurones. Using double-immunofluorescence, glial fibrillary acidic protein-labelled astrocytes and the majority of CART neurones in the ARC showed TRPV5-immunoreactivity. Following iontophoresis of retrograde neuronal tracer, cholera toxin β (CtB) into the anteroventral periventricular nucleus and median preoptic nucleus, retrograde accumulation of CtB was observed in most TRPV5-equipped ARC CART neurones. Next, we determined the response of TRPV5-elements in the ARC during the oestrous cycle. Compared to pro-oestrus, a significant increase (P<.001) in the percentage of TRPV5-expressing CART neurones was observed during oestrus, metoestrus, and dioestrus. TRPV5-immunoreactivity in the astrocytes, however, showed a significant increase during metoestrus and dioestrus. We suggest that the TRPV5 ion channel may serve as an important regulator of neural and neuroendocrine pathways in the brain.

Exercise enhance the ectopic bone formation of calcium phosphate biomaterials in muscles of mice

ObjectiveTo investigate whether exercise can enhance ectopic bone formation of calcium phosphate (Ca-P) biomaterials in muscles of mice. MethodsFirstly, ten transient receptor potential vanilloid subfamily member 1 (TRPV1) knockout mice (group KO) and ten C57BL/6 mice (group WT) were randomly chosen, 10μg Ca-P biomaterials were implanted into the thigh muscle pouch of each mouse which was far away from femur; after that, all animals were kept in open field for free exploration 5min, and the movement time and distance were automatically analyzed. Ten weeks later, the Ca-P samples were harvested for histological staining and immunochemistry. Secondly, the Ca-P biomaterials were implanted into the thigh muscle pouch of C57BL/6 mice the same as previous operation, and then randomly divided into two groups: running group and non-running group (n=10); in running group, all mice run 1h as a speed of 6m/h in a treadmill for 10weeks. Ten weeks later, the blood was collected to detect the interleukin-4 (IL-4) and IL-12 levels by enzyme linked immunosorbent assay (ELISA), and the samples were harvested for histological staining. ResultsIn groups KO and WT, both the movement time and distance were significant higher in group KO than that in group WT (P<0.05); furthermore, the histology staining results showed that lots of new bone and bone marrow tissues were observed in group KO, but only bone matrix-like substances were observed in group WT. In running group and non-running group, the ELISA results indicated that the immunological genes, both IL-4 and IL-12 levels were significant higher in running group than that in non-running group (P<0.05); besides that, more new bone tissues were observed in running group than that in non-running group. ConclusionKnockout of TRPV1 in mice could reduce algesia and induce the stronger athletic ability of mice, causing better osteoinductivity of Ca-P biomaterials both in TRPV1−/− mice and running mice; according to this, we want to offer a proposal to patients who suffer from bone defects and artificial bone transplantation: do moderate exercise, don't convalesce all the time.

TRPV1 activation counters diet-induced obesity through sirtuin-1 activation and PRDM-16 deacetylation in brown adipose tissue.

Background/ObjectiveAn imbalance between energy intake and expenditure leads to obesity. Increasing metabolism and thermogenesis in brown adipose tissue (BAT) can help in overcoming obesity. Here, we investigated the effect of activation of transient receptor potential vanilloid subfamily 1 (TRPV1) in the upregulation of thermogenic proteins in BAT to counter diet-induced obesity.Subjects/MethodsWe investigated the effect of dietary supplementation of capsaicin (TRPV1 agonist) on the expression of metabolically important thermogenic proteins in BAT of wild type and TRPV1−/− mice that received either a normal chow or high fat (± capsaicin; TRPV1 activator) diet by immunoblotting. We measured the metabolic activity, respiratory quotient and BAT lipolysis.ResultsCAP antagonized high fat diet (HFD)-induced obesity without decreasing energy intake in mice. HFD suppressed TRPV1 expression and activity in BAT and CAP countered this effect. HFD feeding caused glucose intolerance, hypercholesterolemia and decreased the plasma concentration of glucagon like peptide-1 and CAP countered these effects. HFD suppressed the expression of metabolically important thermogenic genes, ucp-1, bmp8b, sirtuin 1, pgc-1α and prdm-16 in BAT and CAP prevented this effect. CAP increased the phosphorylation of sirtuin 1 and induced an interaction between PPARγ with PRDM-16. Further, CAP treatment, in vitro, decreased the acetylation of PRDM-16, which was antagonized by inhibition of TRPV1 by capsazepine, chelation of intracellular Ca2+ by cell permeable BAPTA-AM or the inhibition of SIRT-1 by EX 527. Further, CAP supplementation, post HFD, promoted weight loss and enhanced the respiratory exchange ratio. CAP did not have any effect in TRPV1−/− mice.ConclusionsOur data show that activation of TRPV1 in BAT enhances the expression of SIRT-1, which facilitates the deacetylation and interaction of PPARγ and PRDM-16. These data suggest that TRPV1 activation is a novel strategy to counter diet-induced obesity by enhancing metabolism and energy expenditure.

Primary sensory neuron-specific interference of TRPV1 signaling by AAV-encoded TRPV1 peptide aptamer attenuates neuropathic pain.

BackgroundTRPV1 (transient receptor potential vanilloid subfamily member 1) is a pain signaling channel highly expressed in primary sensory neurons. Attempts for analgesia by systemic TRPV1 blockade produce undesirable side effects, such as hyperthermia and impaired heat pain sensation. One approach for TRPV1 analgesia is to target TRPV1 along the peripheral sensory pathway.ResultsFor functional blockade of TRPV1 signaling, we constructed an adeno-associated virus (AAV) vector expressing a recombinant TRPV1 interfering peptide aptamer, derived from a 38mer tetrameric assembly domain (TAD), encompassing residues 735 to 772 of rat TRPV1, fused to the C-terminus of enhanced green fluorescent protein (EGFP). AAV-targeted sensory neurons expressing EGFP-TAD after vector injection into the dorsal root ganglia (DRG) revealed decreased inward calcium current and diminished intracellular calcium accumulation in response to capsaicin, compared to neurons of naïve or expressing EGFP alone. To examine the potential for treating neuropathic pain, AAV-EGFP-TAD was injected into fourth and fifth lumbar (L) DRGs of rats subjected to neuropathic pain by tibial nerve injury (TNI). Results showed that AAV-directed selective expression of EGFP-TAD in L4/L5 DRG neuron somata, and their peripheral and central axonal projections can limit TNI-induced neuropathic pain behavior, including hypersensitivity to heat and, to a less extent, mechanical stimulation.ConclusionSelective inhibition of TRPV1 activity in primary sensory neurons by DRG delivery of AAV-encoded analgesic interfering peptide aptamers is efficacious in attenuation of neuropathic pain. With further improvements of vector constructs and in vivo application, this approach might have the potential to develop as an alternative gene therapy strategy to treat chronic pain, especially heat hypersensitivity, without complications due to systemic TRPV1 blockade.

Transient receptor potential vanilloid 6 (TRPV6) in the mouse brain: Distribution and estrous cycle-related changes in the hypothalamus

Transient receptor potential vanilloid (TRPV) subfamily of cationic channels have emerged as novel players in neural regulation. Unlike other members of TRPV subfamily, TRPV5 and TRPV6 are highly Ca2+-selective. Although TRPV5/TRPV6 transcripts are expressed in mouse brain, understanding the full functional spectrum of these ion channels in the brain is however limited due to the lack of information on their neuroanatomical distribution. We have studied TRPV6 in mouse brain in further detail. In the hypothalamus, while Western blot analysis using TRPV6 specific antiserum showed a distinct ∼95 kDa band corresponding to the molecular weight of TRPV6, transcripts for TRPV6 were detected with RT-PCR. TRPV6-immunoreactive cells/fibers were observed in vascular organ of the lamina terminalis, olfactory bulb, amygdala, hippocampus, septohypothalamic, supraoptic, arcuate (ARC), dorsomedial, and subincertal nuclei. TRPV6-immunoreactive cells/fibers were also observed in the brainstem and cerebellum. Estrogen has emerged as a potential regulator of TRPV6 in peripheral tissues. TRPV6 gene promoter contains estrogen-response element, estrogen activates TRPV6 via estrogen receptor alpha (ERα), and ERα-expressing ARC neurons in mediobasal hypothalamus (MBH) serve as primary site for estradiol feedback. Using double immunofluorescence, co-expression of TRPV6 and ERα was observed in several ARC neurons. MBH of mice during different phases of estrous cycle were subjected to Western blot analysis of TRPV6. Compared to proestrus, a significant reduction (P<0.01) in intensity of TRPV6-immunoreactive band was observed in MBH during metestrus and diestrus phases. While the wide distribution of TRPV6-expressing elements in the brain suggests its role in a range of CNS functions, the ion channel may serve as novel component of the neural pathway mediating effects of estradiol in MBH.

OS 36-09 DEFICIENCY OF THERMOGENESIS AND HEAT SENSATION ACCELERATES OBESITY AND HIGH BLOOD PRESSURE

Objective: Obesity is global healthy problem. Uncoupling protein 1 (UCP1) mainly expresses on brown adipose tissue (BAT), uncouples energy substrate oxidation from mitochondrial ATP production and results in the loss of potential energy as heat. Transient receptor potential vanilloid subfamily, member 1 (TRPV1) is the heat-gated cation ion channel. The aim of this study was to investigate the in vivo role of TRPV1 and UCP1 co-action in obesity. Design and Method: We generated and characterized UCP1−/− mice lacking TRPV1 activity (TRPV1−/−/UCP1−/− mice). The core-body temperature and the locomotor activity were simultaneously measured by the telemetry system. The rate of mitochondrial respiration was measured using the Oxygraph-2k. Cytosol superoxide anion was determined by DHE and mitochondrial superoxide anion was measured by MitoSOX. The levels of ATP in primary cultured brown adipocytes were measured using a firefly luciferase-based ATP assay kit. Primary brown adipocytes were cultured and the fluorescence measurements of cells were made using a fluorescent plate reader. Results: TRPV1 and UCP1 double knockout would appear obesity in advance and more severe comparing with WT, TRPV1−/− and UCP1−/− mice. TRPV1 and UCP1 double knockout in mice reduced functional BAT generation to cause BAT white, and decreased lipolysis comparing with the other three groups. TRPV1−/−/UCP1−/− mice significantly decreased resting oxygen consumptions, heat production and locomotor activities. Mitochondrial respiratory function seriously disordered in BAT of TRPV1−/−/UCP1−/− mice. TRPV1 and UCP1 double knockout resulted in dramatically reduced Ca2+mito uptake in low [Ca2+]cyto that was coupled with H+mito extrusion, which affected ATP generation and oxidative stress in BAT mitochondria. TRPV1 knockout caused to a Ca2+mito uptake compensatory rise. Conclusions: The results of our genetic manipulation studies provide in vivo evidence for the pathogenesis of TRPV1 and UCP1 double knockout in mice caused obesity and offer a novel target to manipulate obesity and associated complications.

Systemic QX-314 Reduces Bone Cancer Pain through Selective Inhibition of Transient Receptor Potential Vanilloid Subfamily 1-expressing Primary Afferents in Mice.

The aim of this study was to determine whether systemic administration of QX-314 reduces bone cancer pain through selective inhibition of transient receptor potential vanilloid subfamily 1 (TRPV1)-expressing afferents. A mouse model of bone cancer pain was used. The authors examined the effects of bolus (0.01 to 3 mg/kg, n = 6 to 10) and continuous (5 mg kg h, n = 5) administration of QX-314 on both bone cancer pain-related behaviors and phosphorylated cyclic adenosine monophosphate response element-binding protein expression in dorsal root ganglion neurons (n = 3 or 6) and the effects of ablation of TRPV1-expressing afferents on bone cancer pain-related behaviors (n = 10). The numbers of flinches indicative of ongoing pain in QX-314-treated mice were smaller than those in vehicle-treated mice at 10 min (3 mg/kg, 4 ± 3; 1 mg/kg, 5 ± 3 vs. 12 ± 3; P < 0.001; n = 8 to 9), 24 h (3 ± 2 vs. 13 ± 3, P < 0.001), and 48 h (4 ± 1 vs. 12 ± 2, P < 0.001; n = 5 in each group) after QX-314 administration, but impaired limb use, weight-bearing including that examined by the CatWalk system, and rotarod performance indicative of movement-evoked pain were comparable. QX-314 selectively inhibited the increase in phosphorylated cyclic adenosine monophosphate response element-binding protein expression in TRPV1-positive, but not in TRPV1-negative, dorsal root ganglion neurons compared to that in the case of vehicle administration (32.2 ± 3.0% vs. 52.6 ± 5.9%, P < 0.001; n = 6 in each group). Ablation of TRPV1-expressing afferents mimicked the effects of QX-314. This study showed that systemic administration of QX-314 in mice inhibits some behavioral aspects of bone cancer pain through selective inhibition of TRPV1-expressing afferents without coadministration of TRPV1 agonists.

Structural and Functional Interactions between Transient Receptor Potential Vanilloid Subfamily 1 and Botulinum Neurotoxin Serotype A.

BackgroundBotulinum neurotoxins are produced by Clostridium botulinum bacteria. There are eight serologically distinct botulinum neurotoxin isoforms (serotypes A–H). Currently, botulinum neurotoxin serotype A (BoNT⁄A) is commonly used for the treatment of many disorders, such as hyperactive musculoskeletal disorders, dystonia, and pain. However, the effectiveness of BoNT⁄A for pain alleviation and the mechanisms that mediate the analgesic effects of BoNT⁄A remain unclear. To define the antinociceptive mechanisms by which BoNT/A functions, the interactions between BoNT⁄A and the transient receptor potential vanilloid subfamily 1 (TRPV1) were investigated using immunofluorescence, co-immunoprecipitation, and western blot analysis in primary mouse embryonic dorsal root ganglion neuronal cultures.Results1) Three-week-old cultured dorsal root ganglion neurons highly expressed transient TRPV1, synaptic vesicle 2A (SV2A) and synaptosomal-associated protein 25 (SNAP-25). SV2A and SNAP-25 are the binding receptor and target protein, respectively, of BoNT⁄A. 2) TRPV1 colocalized with both BoNT⁄A and cleaved SNAP-25 when BoNT⁄A was added to dorsal root ganglia neuronal cultures. 3) After 24 hours of BoNT⁄A treatment (1 nmol⁄l), both TRPV1 and BoNT⁄A positive bands were detected in western blots of immunoprecipitated pellets. 4) Blocking TRPV1 with a specific antibody decreased the cleavage of SNAP-25 by BoNT⁄A.ConclusionBoNT/A interacts with TRPV1 both structurally and functionally in cultured mouse embryonic dorsal root ganglion neurons. These results suggest that an alternative mechanism is used by BoNT⁄A to mediate pain relief.

New Mechanism of Bone Cancer Pain: Tumor Tissue-Derived Endogenous Formaldehyde Induced Bone Cancer Pain via TRPV1 Activation.

In recent years, our serial investigations focused on the role of cancer cells-derived endogenous formaldehyde in bone cancer pain. We found that cancer cells produced formaldehyde through demethylation process by serine hydroxymethyltransferase (SHMT1 and SHMT2) and lysine-specific histone demethylase 1 (LSD1). When the cancer cells metastasized into bone marrow, the elevated endogenous formaldehyde induced bone cancer pain through activation on the transient receptor potential vanilloid subfamily member 1 (TRPV1) in the peripheral nerve fibers. More interestingly, TRPV1 expressions in the peripheral fibers were upregulated by the local insulin-like growth factor I (IGF-I) produced by the activated osteoblasts. In conclusion, tumor tissue-derived endogenous formaldehyde induced bone cancer pain via TRPV1 activation.

Functional expression of TRPV channels in T cells and their implications in immune regulation.

The importance of Ca(2+) signalling and temperature in the context of T cell activation is well known. However, the molecular identities of key players involved in such critical regulations are still unknown. In this work we explored the endogenous expression of transient receptor potential vanilloid (TRPV) channels, a group of thermosensitive and non-selective cation channels, in T cells. Using flow cytometry and confocal microscopy, we demonstrate that members belonging to the TRPV subfamily are expressed endogenously in the human T cell line Jurkat, in primary human T cells and in primary murine splenic T cells. We also demonstrate that TRPV1- and TRPV4-specific agonists, namely resiniferatoxin and 4α-phorbol-12,13-didecanoate, can cause Ca(2+) influx in T cells. Moreover, our results show that expression of these channels can be upregulated in T cells during concanavalin A-driven mitogenic and anti-CD3/CD28 stimulated TCR activation of T cells. By specific blocking of TRPV1 and TRPV4 channels, we found that these TRPV inhibitors may regulate mitogenic and T cell receptor mediated T cell activation and effector cytokine(s) production by suppressing tumour necrosis factor, interleukin-2 and interferon-γ release. These results may have broad implications in the context of cell-mediated immunity, especially T cell responses and their regulations, neuro-immune interactions and molecular understanding of channelopathies.

Molecular and topological membrane folding determinants of transient receptor potential vanilloid 2 channel

Transient Receptor Potential (TRP) channels are related to adaptation to the environment and somatosensation. The transient receptor potential vanilloid (TRPV) subfamily includes six closely evolutionary related ion channels sharing the same domain organization and tetrameric arrangement in the membrane.In this study we have characterized biochemically TRPV2 channel membrane protein folding and transmembrane (TM) architecture. Deleting the first N-terminal 74 residues preceding the ankyrin repeat domain (ARD) show a key role for this region in targeting the protein to the membrane. We have demonstrated the co-translational insertion of the membrane-embedded region of the TRPV2 and its disposition in biological membranes, identifying that TM1-TM4 and TM5-TM6 regions can assemble as independent folding domains. The ARD is not required for TM domain insertion in the membrane. The folding features observed for TRPV2 may be conserved and shared among other TRP channels outside the TRPV subfamily.

Impact of a folic acid-enriched diet on urinary tract function in mice treated with testosterone and estradiol.

Aging men are susceptible to developing lower urinary tract symptoms, but the underlying etiology is unknown and the influence of dietary and environmental factors on them is unclear. We tested whether a folic acid-enriched diet changed urinary tract physiology and biology in control male mice and male mice with urinary dysfunction induced by exogenous testosterone and estradiol (T+E2), which mimics changing hormone levels in aging humans. T+E2 treatment increased mouse urine output, time between voiding events, and bladder capacity and compliance. Consumption of a folic acid-enriched diet moderated these changes without decreasing prostate wet weight or threshold voiding pressure. One potential mechanism for these changes involves water balance. T+E2 treatment increases plasma concentrations of anti-diuretic hormone, which is offset at least in part by a folic acid-enriched diet. Another potential mechanism involves neural control of micturition. The folic acid-enriched diet, fed to T+E2-treated mice, increased voiding frequency in response to intravesicular capsaicin infusion and increased mRNA abundance of the capsaicin-sensitive cation channel transient receptor potential vanilloid subfamily member 1 (Trpv1) in L6 and S1 dorsal root ganglia (DRG) neurons. T+E2 treatment and a folic acid-enriched diet also modified DNA methylation, which is capable of altering gene expression. We found the enriched diet increased global DNA methylation in dorsal and ventral prostate and L6 and S1 DRG. Our results are consistent with folic acid acting to slow or reverse T+E2-mediated alteration in urinary function in part by normalizing water balance and enhancing or preserving afferent neuronal function.

L596–W733 bond between the start of the S4–S5 linker and the TRP box stabilizes the closed state of TRPV4 channel

Unlike other cation channels, each subunit of most transient receptor potential (TRP) channels has an additional TRP-domain helix with an invariant tryptophan immediately trailing the gate-bearing S6. Recent cryo-electron microscopy of TRP vanilloid subfamily, member 1 structures revealed that this domain is a five-turn amphipathic helix, and the invariant tryptophan forms a bond with the beginning of the four-turn S4-S5 linker helix. By homology modeling, we identified the corresponding L596-W733 bond in TRP vanilloid subfamily, member 4 (TRPV4). The L596P mutation blocks bone development in Kozlowski-type spondylometaphyseal dysplasia in human. Our previous screen also isolated W733R as a strong gain-of-function (GOF) mutation that suppresses growth when the W733R channel is expressed in yeast. We show that, when expressed in Xenopus oocytes, TRPV4 with the L596P or W733R mutation displays normal depolarization-induced activation and outward rectification. However, these mutant channels have higher basal open probabilities and limited responses to the agonist GSK1016790A, explaining their biological GOF phenotypes. In addition, W733R current fails to inactivate during depolarization. Systematic replacement of W733 with amino acids of different properties produced similar electrophysiological and yeast phenotypes. The results can be interpreted consistently in the context of the homology model of TRPV4 molecule we have developed and refined using simulations in explicit medium. We propose that this bond maintains the orientation of the S4-S5 linker to keep the S6 gate closed. Further, the two partner helices, both amphipathic and located at the polar-nonpolar interface of the inner lipid monolayer, may receive and integrate various physiological stimuli.

Acidic Extracellular Microenvironment in Myeloma-Colonized Bone Contributes to Bone Pain

Multiple myeloma (MM) bone disease results in devastating bone pain and fractures, which are the major cause of morbidity, and contribute to increased mortality and diminish the quality of life in MM patients. Current treatments for MM bone pain do not completely control the pain and have serious side effects. Thus, new therapies are needed to control myeloma bone pain. However, the mechanisms responsible for MM-associated bone pain are poorly understood. Specific osteoclast inhibitors (bisphosphonates and denosumab) can reduce bone pain in MM patients, suggesting that factors released at the tumor-bone interface during osteoclastic bone resorption may be important contributors to bone pain.Bone-resorbing osteoclasts release protons to dissolve bone minerals and aggressively proliferating tumor cells also release protons/lactate as a consequenece of aerobic glycolysis. Since acidosis is algogenic for primary afferent sensory neurons, we reasoned that an acidic extracellular microenvironment created by the release of protons by osteoclasts and MM cells play a critical role in the pathophysiology of bone pain.To investigate the mechanism of bone pain associated with MM, we used an animal model in which the JJN3 human MM cells were inoculated into the tibiae of SCID mice. Control mice received PBS. Bone pain was assessed by determining tactile hypersensitivity and thermal hyperalgesia of JJN3-bearing mice using von Frey and plantar tests.Administration of the pH probe acridine orange to JJN3-bearing mice revealed that resorption pits beneath bone-resorbing osteoclasts and JJN3-colonized tibiae are acidic. Mice bearing JJN3 MM in their tibiae displayed progressive tactile hypersensitivity and thermal hyperalgesia as the tumor grew. Of note, the bisphosphonate zoledronic acid significantly reduced the progression of the tactile hypersensitivity and thermal hyperalgesia, suggesting that these nociceptive behaviors of JJN3-bearing mice are due to osteoclast-mediated bone pain. Importantly, the non-selective proton pump inhibitor bafilomycin A1 inhibited the creation of the acidic extracellular microenvironment in JJN3-colonized bone and significantly prevented the progression of the nociceptive behaviors. These results support that the acidic extracellular microenvironment is responsible for evoking bone pain. Immunohistochemical examination to identify acid-sensing mechanisms present in bone showed that the calcitonin gene-related peptide (CGRP)-positive sensory neurons innervating bone are adjacent to osteoclasts with co-expression of the acid-sensing nociceptor, the transient receptor potential vanilloid subfamily member 1 (TRPV1). To determine the role of TRPV1 in the excitation of sensory neurons, primary sensory neuron cells isolated from dorsal root ganglion (DRG) were exposed to acidic medium (pH 6.5) and examined for intracellular Ca2+ mobilization using Fura 2 AM calcium imaging assays. Acidic medium induced Ca2+ influx in DRG sensory neuron cells and the induced Ca2+ influx was blocked in the presence of the specific TRPV1 antagonist SB366791, while the specific TRPV4 antagonist RN1734 showed no effect. Further, the specific antagonist for the acid-sensing ion channel 3 (ASIC3), APETx2, also blocked the induction of Ca2+ influx. Neutral medium (pH 7.4) did not induce Ca2+ influx. Taken together, these results suggest that TRPV1 and ASIC3 play an important role in the excitation of sensory neurons exposed to acidic extracellular microenvironment.Our results suggest that the acidic extracellular microenvironments created by protons released from osteoclasts and MM cells excite sensory neurons associated with bone via the acid-sensing nociceptors, TRPV1 and ASIC3, to evoke bone pain. TRPV1 and ASIC3 may be potential targets for ameliorating bone pain in MM. DisclosuresRoodman:Eli Lilly and Co.: Research Funding.

ME-06 * ENDOGENOUS NEURAL PRECURSOR CELLS INDUCE CELL DEATH OF HIGH-GRADE ASTROCYTOMAS AND HAVE A TROPISM TO TUMORS IN THE HUMAN BRAIN

Data from mouse models suggest that high-grade gliomas (HG-gliomas) origin from somatic mutant neural stem and precursor cells (NPCs). We found that endogenous NPCs also defend the brain against HG-gliomas. We observed that NPCs migrate from the subventricular zone to HG-gliomas in mice and we found that NPCs, which are genetically distinct from the tumor mass, accumulate at the border of high-grade gliomas in humans. The vascular endothelial growth factor pathway controls the tropism of mouse and human NPCs to gliomas. Tumour associated NPCs reduce glioma expansion and prolong survival by releasing a group of fatty acid ethanolamides that have agonistic activity on the vanilloid receptor (transient receptor potential vanilloid subfamily member-1; TRPV1). TRPV1 expression is much higher in HG-gliomas than in tumor-free brain and TRPV1 stimulation triggers tumor cell death. NPC-mediated tumor suppression can be mimicked by systemic administration of synthetic vanilloids and phytovanilloids suggesting that TRPV1 agonists hold potential as new HG-astrocytoma therapeutics. Finally, our data reveal that brain tumour suppression is an intrinsic feature of NPCs, but not other stem and precursor cells. Factors secreted from induced pluripotent stem cells (iPS cells) did not trigger glioma cell death, but when iPS cells were differentiated into NPCs they released anti-tumourigenic vanilloids. Overall, our data reveal that NPCs are attracted to high-grade gliomas in mice and humans, that NPCs have a specific role in suppressing HG-gliomas and that NPC-released anti-tumorigenic factors can be used as pre-clinical HG-glioma therapeutics.